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Structural-functional study of the paracellular ion pore

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Structural-functional study of the paracellular ion pore

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Content STRUCTURAL-FUNCTIONAL STUDY OF THE PARACELLULAR
ION PORE

by
Jiahua Li

Dissertation
Presented to the Faculty of the USC Graduate School of
The University of Southern California
in Partial Fulfillment
of the Requirements
for the Degree of

Doctor of Philosophy

The University of Southern California
August 2013

DEDICATION

This dissertation is dedicated to my wife Min Zhuo,
Who embraces me in love and inspires me in pursuit of truth and beauty

iii
ACKNOWLEDGEMENTS
I express my deepest appreciation to my Ph.D advisor, Professor Alan S.L. Yu,
who has established himself as a lifetime mentor for me. He constantly and convincingly
conveyed a spirit of adventure in regard to research and scholarship. His clear and
concise teaching style and inspiring leadership instilled in me an excitement to learn, as
well as a commitment to academic research and leadership. Without his guidance and
persistent mentorship, this dissertation would not have been possible.
I thank my committee members, Professor Alicia A. McDonough, the former
director of the Systems Biology and Disease Program, who creates a comaraderie of the
graduate study and are very supportive to the scholar and career development of trainees.
I also thank Professor Robert A. Farley, Professor Ralf Langen, and Professor Janos Peti-
Peterdi for their inspiring comments and suggestions about the dissertation. Without their
help, this dissertation would take much longer to accomplish.
I express many thanks to my lab managers, Alice Kanzawa and Kayleigh
Peterson. Their detail-oriented lab management constitutes the cornerstone of my work. I
also thank my lab mates: Mien Nguyen, Morten Buch Engelund, Tamako Nakamura, Min
Zhuo, Lei Pei, Madhumitha Rajagopal, Igor Boulatnikov, and Lynn Magenheimer for
always being supportive. It feels so great having you all to share the every frustration and
excitement of the research. In addition, I thank Susanne Angelow and Anna Linge, whose
work lay down a solid foundation of my dissertation.
Last but not the least, I thank my parents for the unconditional love and support.

iv
STRUCTURAL-FUNCTIONAL STUDY OF THE PARACELLULAR
ION PORE

Jiahua Li M.B.
The University of Southern California, 2013

Supervisor: Alan S.L. Yu

Abstract: Claudins constitute a family of tight junction transmembrane proteins
whose first extracellular loop (ECL1) determines the paracellular permeability and ion
selectivity in epithelia. Claudin-2 forms a paracellular cation pore. We are interested in
the molecular mechanism of ion selectivity of claudin-2 from a structural-functional
perspective. In chapter 2, we explored the role of two highly conserved cysteines in
ECL1 by hypothesizing that these extracellular cysteines are linked by an intramolecular
disulfide bond. We found that the single cysteine mutants can form a claudin-2
homodimer, suggesting that the two conserved cysteines normally form an intramolecular
disulfide bond in wild-type claudin-2. We also found that the disulfide bond is necessary
for pore formation. In chapter 3, we tested the role of a highly conserved aromatic residue
near the pore selectivity filter of claudins by hypothesizing that it contributes to cation
selectivity by cation-pi interaction with the permeating cation. The Y67L mutant showed
reduced cation selectivity compared to wild-type claudin-2 due to the decreased Na
+

permeability, without affecting the Cl
-
permeability. The Y67A mutant enlarged the pore
size and further decreased the charge selectivity due to an increase in Cl
-
permeability.
The Y67F mutant restored the Na
+
permeability, Cl
-
permeability, and pore-size back to
v

wild-type. We conclude that the conserved aromatic residue near the cation pore domain
of claudins contributes to cation selectivity by a dual role of cation-pi interaction and a
luminal steric effect. In chapter 4, we aimed to map out all pore-lining residues of
claudin-2 through comprehensive cysteine-scanning mutagenesis of ECL1. We screened
45 cysteine mutants of the ECL1 in polyclonal MDCK II Tet-off cells and found nine
pore-lining residues. Next, we stably expressed these candidates in monoclonal MDCK I
Tet-off cells for confirmatory studies. The mutants had similar ion permselectivity and
pore size as wild-type claudin-2. Nevertheless the conductance inhibition assay of a panel
of MTS reagents revealed distinct patterns of blockage effect and varying kinetics of
reaction. In conclusion, we identified all pore-lining residues of claudin-2 with distinct
geometrical location. This can be applied to future x-ray crystal structures and molecular
modeling of claudins to further understand the molecular mechanism for paracellular ion
transport.
vi
Table of Contents
List of Tables ...........................................................................................................x

List of Figures ........................................................................................................ xi

Chapter 1: Introduction and Background ..............................................................1

Tight Junction Constitutes Paracellular Barrier In the Epithelia ....................1

Claudins in the Tight Junction Regulate the Paracellular Permeability .........3

General properties of claudins ...............................................................3

Expression studies in epithelial cell lines and knockout studies in mice4

Chimera and mutagenesis studies ..........................................................5

Properties of claudin pore ......................................................................6

Clinical Significance of Understanding the Structure-Function of Claudins .8

Chapter 2: Claudin-2 Pore Function Requires an Intramolecular Disulfide Bond
Between Two Conserved Extracellular Cysteines ........................................10

Abstract .........................................................................................................10

Introduction ...................................................................................................11

Materials and Methods ..................................................................................12

Results ...........................................................................................................15

vii
Single mutants form a claudin-2 homodimer linked by an intermolecular
disulfide bond..............................................................................15

Extracellular conserved cysteines are not required for claudin-2 trafficking
to tight junction. ..........................................................................20

Single cysteine mutation makes the remaining extracellular cysteine
accessible to MTSEA-biotin. ......................................................21

The cation pore function is lost in the serine mutants. ........................24

Discussion .....................................................................................................26

Chapter 3: Conserved Aromatic Residue Confers Cation Selectivity in Claudin-2 and
Claudin-10b ..................................................................................................31

Abstract .........................................................................................................31

Introduction ...................................................................................................32

Materials and Methods ..................................................................................33

Results ...........................................................................................................39

Stable transduction of claudin-2 and claudin-10b mutants in MDCK I Tet-
off cells. .......................................................................................39

In claudin-2, leucine substitution leads to partial loss of cation selectivity
without affecting the pore size. ...................................................41

viii
In claudin-2, alanine substitution abolishes cation selectivity and enlarges
pore size ......................................................................................44

In claudin-2, substitution of another aromatic residue at position 67
partially restores cation selectivity and pore size. ......................45

In claudin-2, the side chain of residue 67 is accessible from the pore
lumen. ..........................................................................................45

F66 is critical for the function of claudin-10b. ....................................49

Discussion .....................................................................................................51

Chapter 4: Comprehensive Cysteine-Scanning Mutagenesis Reveals Claudin-2 Pore
Lining Residues with Distinct Characteristics ..............................................56

Abstract .........................................................................................................56

Introduction ...................................................................................................57

Materials and Methods ..................................................................................58

Results ...........................................................................................................65

Characterization of polyclonal MDCK II Tet-off cell lines expressing each
cysteine mutant. ..........................................................................65

Screening assay of pore conductance inhibition by MTSET identifies
candidates of pore-lining residue. ...............................................70

ix
Biochemical and electrophysiological characterization of monoclonal
MDCK I Tet-off cell lines expressing the pore-lining residues. .72

Pore-lining residues reveal distinct patterns of conductance inhibition by
thiol-reactive reagents. ................................................................76

Kinetics of reaction of MTS reagents with the substituted pore-lining
cysteines. .....................................................................................80

Discussion .....................................................................................................83

Chapter 5: Summary and Future Direction ............................................................88

Summary .......................................................................................................88

Overall Significance of This Dissertation .....................................................89

Future Directions ..........................................................................................89

Claudin-claudin interface .....................................................................90

Structural modeling and stimulation of claudins .................................90

References ..............................................................................................................92

x

List of Tables
Table 1-1. Biological Roles of Claudins in Health and Diseases ............................8

Table 4-1. Biological and Physiological Characterization of 45 Cysteine Mutants of
Claudin-2 ECL1 in Polyclonal MDCK II Tet-off Cells ...................68

Table 4-2. Summary of Estimated Pore Size of Cysteine Mutants and Wild-Type
Claudin-2 ..........................................................................................75

Table 4-3. Size, Charge, and Working Concentration of The Thiol-Reactive Reagents
Used In This Study* .........................................................................77

xi
List of Figures
Figure 1-1. Epithelial cells are connected through junctional complexes. ..............1

Figure 1-2. The composition of tight junction. ........................................................2

Figure 1-3. The first extracellular loop of claudins forms the paracellular pore. ....6

Figure 1-4. Properties of claudin-2 pore. .................................................................7

Figure 2-1. Immunoblot of cell lysates from cell lines stably transduced with wild-
type claudin-2 and claudin-2 mutants. ..............................................17

Figure 2-2. Effect of the reducing agent, 2-mercaptoethanol, on the higher molecular
weight band. ......................................................................................18

Figure 2-3. Claudin composition of the putative dimer band. ...............................19

Figure 2-4. Localization of claudin-2 mutants by immunofluorescent staining and
confocal microscopy examination. ...................................................21

Figure 2-5. Identification of accessible extracellular cysteines in claudin-2 constructs
by MTSEA-biotin biotinylation. .......................................................23

Figure 2-6. Characterization of the electrophysiological properties of claudin-2 serine
mutants. .............................................................................................25

Figure 3-1. Characterization of MDCK I Tet-Off cell lines stably transduced with
claudin-2 and claudin-10b constructs. ..............................................40

Figure 3-2. Characterization of the electrophysiological properties of claudin-2
constructs. .........................................................................................43

Figure 3-3. Characterization of the structural-functional properties of claudin-2
Y67C. ................................................................................................47

Figure 3-4. Conductance inhibition assay by MTSET and MTS-Bn. ...................48

xii
Figure 3-5. Characterization of the electrophysiological properties of claudin-10b
wild-type (WT). ................................................................................49

Figure 3-6. Characterization of the electrophysiological properties of claudin-10b
constructs. .........................................................................................50

Figure 3-7. Homology alignment of major pore-forming claudins. ......................55

Figure 4-1. Screening of protein expression in polyclonal MDCK II Tet-off cells
expressing 45 single cysteine mutants. .............................................66

Figure 4-2. Screening of protein localization in polyclonal MDCK II Tet-off cells
expressing 45 single cysteine mutants. .............................................67

Figure 4-3. Screening assay of conductance inhibition by MTSET. .....................72

Figure 4-4. Characterization of stably transfected MDCK I Tet-Off cell lines
expressing claudin-2 mutants. ...........................................................73

Figure 4-5. Characterization of the electrophysiological properties of claudin-2
cysteine mutants of the pore-lining residues. ....................................74

Figure 4-6. Conductance inhibition reflects pore location. ....................................76

Figure 4-7. Confirmatory assay of conductance inhibition by a panel of thiol-reactive
reagents. ............................................................................................79

Figure 4-8. Kinetics of reaction reflects pore location. .........................................81

Figure 4-9. Kinetics of reaction of MTS reagents with the substituted cysteines. 82

Figure 4-10. Speculative model of pore region. ....................................................87

1
CHAPTER 1: INTRODUCTION AND BACKGROUND
TIGHT JUNCTION CONSTITUTES PARACELLULAR BARRIER IN THE
EPITHELIA
Epithelial cells are connected via multiple junctional complexes. The tight
junction separates the apical and basolateral membrane domains and acts as the
paracellular barrier while remaining selectively permeable to ions and water. By electron
microscopy, the tight junction appears as a band of parallel fibril strands in the subapical
compartment. Ussing and Windhager (54) incubated frog skin cells apically with Ba
2+

and basolaterally with SO
4
2-
and showed that, under the condition of hyperosmotic
solution on the apical side, BaSO
4
precipitated at the tight junction. Machen (35)
demonstrated that La
3+
permeates the junctional complex in rabbit gallbladder and ileum
epithelium and proposed the concept that some tight junctions are "leaky" in terms of
permeability to ion and small molecule like water and mannitol.

Figure 1-1. Epithelial cells are connected through
junctional complexes.
These include, from the apical to the basolateral side,
tight junction, adherens junction, desmosome, and gap
junction. Tight junction seals the paracellular space
and functions as the paracellular barrier while remains
selective permeable to water and electrolytes.

2
Studies on the biochemical composition of the tight junction have helped to
elucidate the regulation of tight junction permeability. The tight junction is constituted of
several groups of proteins (Figure 1-2). The first group consists of membrane-spanning
proteins (claudin, occudin, and junctional adhesion molecules), which are of particular
interest because their extracellular domains face the paracellular space and are thought to
regulate paracellular transport directly. The second group consists of scaffolding proteins
(e.g. zona occludens family), which link the membrane-spanning proteins to the actin
cytoskeleton. The third group consists of signaling molecules, including transcriptional
factors and kinases/phosphatases that regulate tight junction protein transcription and
expression (for a more detailed review, see (20)).

Figure 1-2. The composition of tight junction.
Tight junction consists of three groups of proteins. The first group is transmembrane protein, including
claudin, occludin, and junctional adhesion molecules, whose extracellular domain protrudes to the
paracellular space. The second group is scaffold proteins, including ZO-1, ZO-2, and ZO-3, which bind
to the intracellular domain of the transmembrane protein and anchor them to the actin cytoskeleton. The
third group (not shown in the figure) is regulatory protein, including transcriptional factors, kinases, and
phosphatases, which regulate the function, localization and expression of the transmembrane proteins.

3
CLAUDINS IN THE TIGHT JUNCTION REGULATE THE PARACELLULAR
PERMEABILITY
General properties of claudins
Twenty-seven claudin genes have so far been indentified in mammalian cells.
Claudins are tetra-transmembrane domain proteins that have two extracellular loops
facing the paracellular space. Among the membrane-spanning proteins at the tight
junction, claudins are by far the most variable in terms of the number of genes/isoforms.
These isoforms are expressed variably in epithelia of different organs in a tissue-specific
manner, which probably correlates with the variability in permeability of these epithelia.
The initial discovery of the pathogenic linkage of claudin-16 to familial hypomagnesemia
hypercalciuria nephrocalcinosis (FHHNC) first suggested that the claudins might be
general regulators of paracellular permeability at the tight junction. At the tight junction,
claudins work in concert with the other components of the tight junction, which makes it
difficult to control for and define the specific roles in permeability of any individual
claudin. Nevertheless, a variety of experimental approaches have been used to answer
three key questions: what is the permeability properties of a specific claudin? What part
of the claudin determines the permeability? What amino acids are the ion binding-sites of
the pore? These studies are summarized in the following section.

4
Expression studies in epithelial cell lines and knockout studies in mice
The function of claudins has been addressed by numerous overexpression studies
in epithelial cell lines (for a detailed review, see (3)). To briefly summarize, claudin-1
(36), claudin-5 (59), claudin-8 (64), claudin-9 (41), claudin-11 (55), and claudin-19 (2)
are considered to be barrier claudins because they increase transepithelial resistance
(TER) when overexpressed in cell lines of low TER. Claudin-2 (17), claudin-4 (26),
claudin-10a (58), claudin-10b (58), claudin-15 (51), 16 (23), claudin-17 (33) are
considered to be pore claudins because they decrease the TER in most cell lines. Because
epithelial cells express endogenous claudins, the change of TER and ion selectivity
reflects the overall effect of exogenous and endogenous claudins interaction rather than
the properties of the expressed claudin alone.
The function of claudins has also been addressed by knockout studies in mice.
This study design minimizes the claudins interaction effects and may better reveal the
function of the target claudin of interest. Claudins-1 knockout (KO) mice and claudin-5
KO mice have an increase of paracellular permeability in the skin and blood-brain
barrier, respectively. Knockout of claudin-2 (40) and claudin-16 (27) decrease
permeability in the proximal renal tubule and in the thick ascending limb of Henle
respectively, while knockout of claudin-7 causes profound renal salt wasting and
dehydration (52). Taken together, these findings confirm that claudins regulate
paracellular permeability at the tight junction.

5
Chimera and mutagenesis studies
While the previous studies showed that claudins regulate the paracellular
permeability, evidence that claudins form the paracellular pore/barrier themselves came
from chimera and mutagenesis studies (Figure 1-3). As mentioned above, claudins have
two extracellular loops. Colegio et al., (6) swapped the extracellular loops between
claudin-2 (a cation pore claudin) and claudin-4 (a cation barrier claudin) and showed that
the first extracellular loop (ECL1) of claudin-2 with the second extracellular loop (ECL2)
of claudin-4 still functioned as a cation pore, whereas the ECL1 of claudin-4 with the
ECL2 of claudin-2 functioned as a cation barrier. Thus, it directly supports the theory that
claudins form either a paracellular pore or a barrier and that this is determined by the
ECL1. Taking this a step further, mutagenesis studies of the ECL1 of claudins revealed
the existence of sites conferring permeability and selectivity. Using charge-reversing
mutations, Colegio (7) first showed that K65D (positive charge mutated to negative
charge) in claudin-4 increased Na
+
permeability, while D55R and E64K (negative charge
mutated to positive charge) both individually and synergistically reversed the claudin-15
ion preference from Na
+
to Cl
-
. However, the effect of reversing the charge at a site may
not necessarily reveal the normal physiological role of the residue at that site but could
instead reflect non-physiological effects of introducing the opposite charge. Yu, el al.
(63) therefore made charge-neutralizing mutations in claudin-2. They found that D65
appears to be an intrapore Na
+
binding site. In aggregate, these data support a model in

6
which the ECL1 of claudins forms the lining of the paracellular pore and determines the
ions permselectivity of paracellular transport.

Properties of claudin pore
The claudin pore is a narrow, fluid-filled, charge-selective, and size-selective pore
formed by the first extracellular loop of adjacent claudins. Ions are transported passively,
parallel to the cell membrane within the paracellular space, driven by the chemical and
electrical gradients. The estimated diameter of claudin-2 pore is approximately 6.5-7.5 Å
(56, 63). An aspartic acid 65 is the intrapore ion-binding site and confers cation
selectivity (63). The pore is a homotypic multimeric complex formed by cis- and/or trans-
Figure 1-3. The first extracellular loop of claudins forms the paracellular pore.
Cation barrier-forming claudin is colored in red and the cation pore-forming claudin is colored in green.
Their first extracellular loop (ECL1) protrudes into the paracellular space. On the left panel, the ECL1
of a pore-forming claudin was swapped into a barrier-forming claudin, and this transform the barrier
claudin into a pore claudin. On the right panel, the ECL1 of a barrier-forming claudin was swapped into
a pore-forming claudin, and this transform the pore claudin into a barrier claudin. This suggests the
ECL1 directly forms the paracellular barrier/pore.

7
interaction between adjacent claudin-2 molecules (8, 18). The pioneer work of selective
cysteine mutagenesis (5) has identified another non-charged residue (I66) lining the pore,
as well as residues located at the intermolecular interface, which prompts us to pursue a
comprehensive cysteine-scanning mutagenesis study to completely map out each residue
in ECL1 and their roles in claudin-2 pore (Figure 1-4).

Figure 1-4. Properties of claudin-2 pore.
The first extracellular loop (ECL1) of claudin-2 forms the paracellular cation pore. The pore is formed
by the homotypic head-to-head interaction of ECL1s on two connecting cells, as well as the homotypic
side-by-side interaction of ECL1s on the same tight junction strand within one cell. Aspartic acid 65
(D65) is the intrapore ion-binding site that confers cation selectivity. There are two conserved cysteines
and their role will be discussed in Ch.2.

8
CLINICAL SIGNIFICANCE OF UNDERSTANDING THE STRUCTURE-
FUNCTION OF CLAUDINS
Claudins have broad functions in the epithelia (Table 1-1). Claudin targeted drugs
can be used in treating diarrhea in inflammatory bowel disease, or in decreasing sodium
reabsorption to lower blood pressure, or as chemotherapeutic agents to claudins related
epithelial cancer. Specifically in sodium homeostasis, one third of filtrated sodium is
reabsorbed in the renal proximal tubules through the paracellular pathway via the
claudin-2 pore. Understanding the critical regions for proper protein folding and function
is crucial for developing drugs that target the paracellular ion pore. The structure-function
study of claudin-2 pore could accelerate the development of claudin-based therapies.
Table 1-1. Biological Roles of Claudins in Health and Diseases
Category Isoform Biological role
Electrolytes
balance
Claudin-16/19 (24) Ca
2+
and Mg
2+
reabsorption in the thick ascending limb of Henle.
Claudin-7 (52) Salt balance in the distal renal tubule.
Claudin-2 (39) Paracellular reabsorption of salt in the proximal renal tubule.
Nutrients
reabsorption
Claudin-15 (50) Provide a luminal sodium gradient for glucose reabsorption in the
intestine.
Claudin-2/12 (16) Vitamin D dependent calcium reabsorption in intestine.
Epithelial
integrity
Claudin-2 (65) Up-regulated in inflammatory bowel diseases, impairing the barrier
function of the intestine.
Claudin-5 (42) Maintain the blood-brain barrier integrity.

9
Claudin-11 (38) Maintain CNS myelin and the tight junction of Sertoli cell.
Infection Claudin-1/6/9 (15,
66)
Co-receptors for HCV entry into hepatocyte.
Cancers Claudin-3/4
(37)(review)
Up-regulated in many epithelial origin cancers, such as
gastrointestinal, ovarian and breast cancers.

10
CHAPTER 2: CLAUDIN-2 PORE FUNCTION REQUIRES AN
INTRAMOLECULAR DISULFIDE BOND BETWEEN TWO
CONSERVED EXTRACELLULAR CYSTEINES
ABSTRACT
Claudins constitute a family of tight junction transmembrane proteins whose first
extracellular loop (ECL1) determines the paracellular permeability and ion selectivity in
epithelia. There are two cysteines in the ECL1 that are conserved among all claudins. We
hypothesized that these extracellular cysteines are linked by an intramolecular disulfide
bond that is necessary for correct pore folding and function. To test this, we mutated C54
and C64 in claudin-2, either individually or together to alanine or serine, and generated
stable MDCK I Tet-off cell lines. Immunoblotting showed a higher molecular weight
band in the mutants with a single cysteine mutation, consistent with a claudin-2 dimer,
suggesting that the two conserved cysteines normally form an intramolecular disulfide
bond in wild-type claudin-2. By immunofluorescent staining, the alanine mutants were
mislocalized intracellularly, while the serine mutants were expressed at the tight junction.
Thus, dimerization of both C54A and C64A did not require tight junction expression,
suggesting that C54 and C64 are located near an intermolecular interface involved in cis-
interaction. The conductance and Na
+
permeability of the serine mutants were markedly
lower than the wild-type, but there was no difference between the single mutants and the
double mutant. We conclude that the disulfide bond between the conserved extracellular

11
cysteines in claudin-2 is necessary for pore formation, probably by stabilizing the ECL1
fold, but is not required for correct protein trafficking. We further speculate that this role
is generalizable to other claudin family members.
INTRODUCTION
The tight junction complex is the most apical junctional complex in epithelia and
is the main determinant of paracellular permeability. Claudins constitute a family of tight
junction transmembrane proteins whose first extracellular loop (ECL1) protrudes into the
paracellular space and forms the lining of the paracellular pore or barrier. A claudin pore
has an estimated diameter of 6.5Å to 8Å (56, 63) and its charge selectivity is determined
by the electrostatic force exerted by charged amino acid(s) on the ECL1 (26, 33, 58, 63).
However, precisely what determines the correct folding of ECL1 is incompletely
understood.
The claudin family shares a highly conserved signature sequence GLW-[X]
2
-C-
[X]
8-10
-C

in the ECL1. The two cysteines in the ECL1 signature sequence are the only
extracellular cysteines and are completely conserved in all claudins. They are believed to
be important in the folding of ECL1. In claudin-5, a barrier claudin, mutating either one
of the conserved cysteines resulted in lower transepithelial resistance (59). In cells
expressing claudin-1, a co-receptor for cell entry of hepatitis C virus (HCV), mutating the
cysteines caused the cells to become insensitive to HCV infection (10). These studies
suggest that, in general, loss of the conserved cysteines impairs claudin function. In a
previous study of claudin-2, the conserved cysteines, C54 and C64, were not modifiable

12
by thiol-reactive reagents (4). Thus, we hypothesized that C54 and C64 are linked by an
intramolecular disulfide bond that is necessary for correct pore folding and proper
function of claudin-2.
To test this hypothesis, we mutated C54 and C64 in claudin-2, either individually
or together, to alanine or serine, and generated stable MDCK I Tet-off cell lines
expressing each mutant protein. Our data suggest that the two conserved cysteines in the
ECL1 are disulfide bonded and that the disulfide bond is necessary for pore formation,
but not for claudin trafficking.
MATERIALS AND METHODS
Generation and screening of MDCK I Tet-off claudin-2 cell lines. Six MDCK I
Tet-off cell lines expressing claudin-2 mutants (C54A, C64A, C54A/C64A, C54S, C64S,
and C54S/C64S) were generated by the methods described previously (62). In short, the
mutants were generated by site-directed mutagenesis on the template plasmid,
pRevTREP-mouse-claudin2-wild-type, using the QuikChange Kit (Agilent
Technologies). The plasmids were lipofected into the viral packaging cell line, PT67.
Viral particles were collected from the growth medium PT67 cells and used to transduce
MDCK I Tet-off cells. After 7-10 days in a 0.3 mg/ml hygromycin selective medium,
independent clones were selected using cloning cylinders. To induce claudin-2
expression, doxycycline was omitted from the culture medium; otherwise 50 ng/mL
doxycycline was included to suppress the protein expression.

13
Immunoblotting. Claudin-2 protein expression was tested by SDS-PAGE and
immunoblotting. Confluent cells grown on tissue culture dishes were mechanically lysed
by passing through a 25-gauge needle 10 times in a sucrose-histidine lysis buffer,
containing 250mM sucrose, 30mM histidine, 1mM EDTA (pH 7), and protease inhibitor
(Complete Mini, Roche Diagnostics). Cell lysates were loaded in a reducing SDS-PAGE
buffer (1% (v/v) 2-mercaptoethanol added) or a non-reducing SDS-PAGE buffer, and
heated at 75˚C for 10 minutes. 20µg of protein samples were loaded on 12%
polyacrylamide gel, transferred to a PVDF membrane, blotted with 1:500 mouse anti-
claudin-2 antibody or 1:1000 rabbit anti-claudin-2 antibody (Invitrogen) and appropriate
horseradish peroxidase-conjugated secondary antibodies (GE), detected with the
chemiluminescent method (Pierce), and imaged by an ImageQuant LAS-4000 (GE
Healthcare).
Immunofluorescent staining. The MDCK I claudin-2 wild-type and mutants were
plated at a density of 10
5
cells/1.16 cm
2
on 12-well transwell plates and grown for seven
days. The cells were washed in ice-cold phosphate buffered saline (PBS), fixed with 4%
paraformaldehyde (PFA) at 4˚C for 15 minutes, permeabilized and blocked in a
permeation buffer (0.3% Triton X-100, 1% BSA, and 5% goat serum in PBS) for one
hour. The filters were incubated in primary antibodies (rabbit anti-claudin-2, 1:200;
mouse anti-ZO-1, 1:400) for two hours at room temperature, washed three times in PBS,
then incubated in secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG and
Alexa Fluor 555-conjugated anti-mouse IgG, both 1:1000, from Life Technologies) for

14
one hour, washed three times in PBS, and mounted in ProLong anti-fade mounting
medium (Life Technologies). Slides were imaged by a Leica TCS SP2 multi-photon
confocal microscope with Z-sections of 0.4µm thickness per layer.
Cysteine-specific surface biotinylation. To test the accessibility of cysteine,
cysteine-specific biotinylation was performed. Cells were plated at a density of 5x10
5
cells/well on a 6-well plate and grown for six days. Cells were pretreated with 2mM
TCEP with or without 0.01% Tween-20 or 0.005% NP-40 for 30 minutes, washed with
PBS with 1mM CaCl
2
and 1mM MgCl
2
(PBS/CM), and treated with 0.5ml/well freshly
dissolved 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) in PBS/CM at
a concentration of 0.5mg/ml. The plate was incubated at room temperature for 10
minutes, washed three times with ice-cold PBS, and the cells were harvested in RIPA
buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholic
acid, 1% (v/v) NP-40). The cell lysates were centrifuged at 16,000 g for 15 minutes. The
supernatant was added to a 40µl slurry of streptavidin-coated beads and rotated at 4˚C for
two hours. The beads were then pelleted and the supernatant was saved for analysis. The
beads were washed three times in TBS (50mM Tris-HCl and 150mM NaCl), added to
20µl 2x reducing SDS-PAGE loading buffer, and heated at 75˚C for 10 minutes with
occasional agitation. Both the bead (biotinylated protein fraction) and supernatant (non-
biotinylated fraction) samples were then subjected to immunoblotting as described above.
Electrophysiological studies in Ussing chamber. Cells were plated at a density of
10
5
cells/1.16cm
2
on Snapwell filters (Corning) and cultured for seven days in the

15
presence (Dox+) or absence (Dox-) of 50 ng/ml doxycycline. The Ussing chamber setup
and liquid junction potential correction method was employed as previously
described(62). In short, the standard Ringer solution used at baseline contained (in mM):
NaCl 150, CaCl
2
2, MgCl
2
1, glucose 10, Tris-HEPES 10, pH 7.4. To measure Na
+

permeability, the solution of the basolateral chamber was changed to a 75mM NaCl
Ringer solution (osmolarity adjusted with mannitol). The ion permeability ratio, β=P
Cl
-
/P
Na
+
, was calculated from the Goldman-Hodgkin-Katz voltage equation. The absolute
Na
+
permeability was estimated by the method devised by Kimizuka and Koketsu’s (29).
The conductance and permeability attributable to claudin-2 pore was calculated by
subtracting the average value of the uninduced (Dox+) state from the values of the
induced (Dox-) state.
Statistics. The data are presented as mean ± standard error. Statistical significance
was determined using unpaired two-tailed Student’s t-test or one-way ANOVA test. The
P value of the multiple comparisons was corrected using the Bonferroni correction. P<
0.05 was considered to be statistically significant.
RESULTS
Single mutants form a claudin-2 homodimer linked by an intermolecular disulfide
bond.
C54 and C64 were mutated individually (single mutants) or together (double
mutants) to either alanine or serine. The rationale for making both alanine and serine

16
mutants is to avoid misinterpretation of data that is due to the artifact of introducing the
mutation. Each mutant was transduced into MDCK I Tet-off cells using retroviral
transduction and stable clones were selected. Inducible protein expression was verified by
immunoblotting (Figure 2-1), in which all mutants showed a claudin-2 monomer band at
approximately 20kD in the absence of doxycycline. When normalized to β-actin, the
protein expression of the single mutants was similar to wild-type and the double mutants
expressed approximately 2-3 fold more protein than wild-type. There were multiple
smaller molecular weight bands present in the C64 single mutants and the double
mutants, which were not seen in the claudin-2 blot of mouse kidney lysates using the
same antibody (data not shown). These bands therefore are probably products of
proteolysis, which are commonly seen in overexpressing exogenous protein in cells.
Also, the double mutants had multiple higher molecular weight bands, which might be
related to post-translational modification, but they have not been fully characterized yet.
Interestingly, there was an additional higher molecular weight band clearly present in
C64A and C64S, and just visible in C54A and C54S. This higher molecular weight band
was not seen in the wild-type or in the double mutants. The band was approximately
35kD, smaller than the predicted size of a claudin-2 dimer. However, since claudin-2
monomer migrates faster than its predicted size of 24kD, the dimer may migrate faster as
well. In addition, although the putative dimer band was not exactly at the molecular
weight of a claudin-2 dimer (40kD), this band had the same apparent molecular weight as
D65C, which had previously been suggested to form a disulfide-bonded dimer(5). We

17
hypothesized that in wild-type claudin-2, C64 is normally in an intramolecular disulfide
bond with C54. Mutation of C64 to alanine or serine would free the thiol side chain of
C54 to interact with C54 on an adjacent molecule, forming an intermolecular disulfide-
bonded dimer. Likewise, mutating C54 could free up C64 to form an intermolecular
disulfide bond with its counterpart on a neighboring molecule.

To verify this hypothesis, protein samples were loaded with non-reducing SDS-
PAGE buffer and reducing SDS-PAGE buffer. Under non-reducing conditions, the four
single mutants (C54A, C64A, C54S, and C64S) exhibited a higher molecular weight
band at the same level of the dimer band of D65C. Furthermore, under reducing
conditions, the abundance of this band decreased concomitant with the increased
20kD
25kD
37kD
Doxycycline
C54A C64A C54A/C64A WT
+- +- +- -
C54S
+-
C64S
+-
C54S/C64S
+-
WT
-
Claudin-2
-actin
Figure 2-1. Immunoblot of cell lysates from cell lines stably transduced with wild-type claudin-2 and
claudin-2 mutants.
Cells were cultured in the presence (+) or absence (-) of doxycycline. Cell lysates were subjected to
reducing SDS-PAGE and immunoblotted using mouse anti-claudin-2 antibody. Inducible expression
of a band at the predicted molecular weight for claudin-2 monomer at approximately 20kD was seen in
all mutants. Note the appearance of a higher molecular weight band at approximately 35 kD, most
prominently in C64A and C64S in the absence of doxycycline.

18
abundance of the monomer band (Figure 2-2), demonstrating that the band was a
disulfide-bonded dimer and that it was composed of at least one claudin-2 molecule.
There was a residual dimer band of C64S and C64A in reducing conditions either
because the intermolecular disulfide bond formed by C54 is very stable and cannot be
completely reduced, or because its position within the protein fold makes it poorly
accessible to reducing agents.

2-mercaptoethanol
C54A
-+
C64A
-+
C54A/C64A
-+
C54S
-+
C64S
-+
C54S/C64S
-+
D65C
-
WT
-
20kD
30kD
40kD
D65C
-
WT
-
20kD
25kD
37kD
2-mercaptoethanol
Figure 2-2. Effect of the reducing agent, 2-mercaptoethanol, on the higher molecular weight band.
Cell lysates from MDCK I Tet-off cells expressing wild-type claudin-2 and the indicated mutants were
loaded on an SDS-PAGE gel using either non-reducing (-) or reducing (+) gel loading buffer, and
immunoblotted with anti-claudin-2. D65C was included as a positive control to show the expected size
of a claudin-2 dimer. In mutants of a single cysteine, a higher molecular weight band consistent with a
claudin-2 dimer was seen in non-reducing conditions, and the abundance of this band decreased under
reducing conditions. This band was not present in wild-type or in the double cysteine mutants

19
To further confirm that the band was a claudin-2 homodimer, the membrane was
blotted with antibodies against the other claudin isoforms known to be present in MDCK
I cells: claudin-1, claudin-3, claudin-4, and claudin-7 (1, 17, 52, 63). No
heterodimerization of claudin-2 with claudin-3, claudin-4, or claudin-7 was seen. In the
claudin-1 blot, there was a faint band just above 25kD in all samples, consistent with a
nonspecific band. Also, there was another faint band present at approximately 35kD in
C54A and C64A, but not in D65C or wild-type, suggesting a possible heterodimerzation
of claudin-1 and claudin-2 single mutant (Figure. 2-3). However, the abundance of this
band was extremely low compared to the dimers in the claudin-2 blot. We conclude that
the vast majority of the single mutant molecules form claudin-2 homodimers.

20kD
25kD
37kD
C54A C64A D65C WT
IB: Claudin-1 IB: Claudin-2 IB: Claudin-3 IB: Claudin-4 IB: Claudin-7
C54A C64A D65C WT C54A C64AD65C WT C54A C64A D65C WT C54A C64A D65C WT L
15kD
50kD
Figure 2-3. Claudin composition of the putative dimer band.
Cell lysates from MDCK I Tet-off cells expressing C54A, C64A, D65C, and wild-type were loaded on
an SDS-PAGE gel using a non-reducing gel loading buffer and blotted using anti-claudin-1, anti-
claudin-2, anti-claudin-3, anti-claudin-4, and anti-claudin-7 antibodies respectively. The signal of the
other claudins was exposed to similar intensity as the signal of claudin-2.

20
Extracellular conserved cysteines are not required for claudin-2 trafficking to tight
junction.
Mutating conserved amino acids may cause protein misfolding and retention in
the endothelium reticulum. The localization of the mutants was therefore examined by
immunofluorescent staining of the tight junction marker ZO-1 and claudin-2, followed by
confocal microscopy imaging. Initially, we generated the alanine mutants and found that
C54A and C54A/C64A were localized intracellularly, while C64A expressed both
intracellularly and on the lateral membrane. This suggests that the conserved cysteines
may be important for claudin-2 trafficking, but we could not exclude the possibility of an
artifact due to introducing the alanine. Because serine mutations at this site were found to
be well tolerated in claudin-5 (59), we then generated the serine mutants and found that
the serine mutants co-localized with ZO-1 at the tight junction similar to wild-type
(Figure. 2-4). This suggests that the loss of the disulfide bond per se does not affect
claudin-2 trafficking and, by inference, that it probably is not critical for global protein
folding. However, the specific introduction of alanine at positions 54 or 64 may in some
way disrupt normal protein trafficking.

21

Single cysteine mutation makes the remaining extracellular cysteine accessible to
MTSEA-biotin.
If C54 and C64 are normally disulfide-bonded to each other in wild-type claudin-
2, there should be no accessible free thiol groups on the extracellular surface of claudin-2.
Consistent with this prediction, we previously demonstrated that wild-type claudin-2
could not be biotinylated by extracellular addition of the membrane-impermeant thiol-
reactive reagent, MTSEA-biotin (5). As a positive control in this experiment, MTSEA-
biotin was shown to be able to biotinylate a claudin-2 mutant, Y35C, in which a cysteine
was introduced at an extracellularly accessible site. It should be noted that some wild-
Figure 2-4. Localization of claudin-2 mutants by immunofluorescent staining and confocal microscopy
examination.
Cells were cultured on Transwells for six days, then immunostained with ZO-1 (red) and claudin-2
(green) antibodies. A series of Z-sections of 0.4µm thickness per layer was collected for each mutant.
The Z-stack along the y-axis was displayed on the right of the image and the Z-stack along the x-axis
was displayed below the image. All serine mutants co-localized well with ZO-1 at the tight junction.
C54A and C54A/C64A were mislocalized intracellularly. C64A were localized both intracellularly and
on the lateral membrane.

22
type claudin-2 could be recovered in the streptavidin bead fraction even without MTSEA-
biotin (Figure 2-5A), probably due to nonspecific binding of claudin-2 molecules to the
beads. We attempted to reduce the putative intramolecular disulfide bond with a strong
reducing agent, TCEP, either on its own or together with weak detergents to partially
unfold the protein. There was a small fraction of claudin-2 recovered in the bead fraction
of both the wild-type without TCEP and the C54S/C64S samples, probably due to non-
specific binding. Nevertheless, we were unable to detect any enhancement of
biotinylation in TCEP treated groups (Figure 2-5B). This indicates that the disulfide bond
is buried inside the extracellular fold of the protein where it is inaccessible to TCEP.
Our hypothesis further predicts that the mutation of either one of the extracellular
cysteines will free up the other cysteine and makes it biochemically accessible to
covalent modification by MTSEA-biotin. Indeed, we found that both C54S and C64S
could be biotinylated by MTSEA-biotin, whereas there was a very small amount of
biotinylated fraction in wild-type due to non-specific binding and no biotinylated fraction
in C54S/C64S (Figure. 2-5C). This provides additional support for the contention that
C54 and C64 are normally disulfide bonded to each other in the native protein.

23

Figure 2-5. Identification of accessible extracellular cysteines in claudin-2 constructs by MTSEA-
biotin biotinylation.
(A) MDCK I Tet-off cells expressing wild-type claudin-2 were treated with or without MTSEA-biotin,
followed by the biotinylation procedure, and were subjected to SDS-PAGE and blotted by anti-
claudin-2 antibody. (B) MDCK I Tet-off cells expressing wild-type claudin-2 were pre-incubated with
2mM TCEP with or without detergent (0.01% Tween-20 or 0.005% NP-40) for 30 min, and then were
subjected to MTSEA biotinylation and streptavidin precipitation. The beads (B) and the supernatant
(S) were subjected to SDS-PAGE and blotted by anti-claudin-2 antibody. MDCK I Tet-off claudin-2
Y35C and C54S/C64S were included as the positive and negative controls for the extracellular
cysteine biotinylation, respectively. (C) MDCK I Tet-off cells expressing serine mutants were cultured
for six days, and then exposed to 0.5mg/ml MTSEA-biotin. The cells were lysed in RIPA buffer and
then precipitated by streptavidin beads. The beads and the supernatant samples were subjected to SDS-
PAGE and immunoblotted with anti-claudin-2 antibody (both beads and supernatant samples) and
with anti-β-actin (supernatant samples) for loading control.

BS
2mM TCEP
0.01% Tween-20
0.005% NP-40
+
WT
++ - - - - -
-+ - - + - - -
-- + - - + - -
BS
WT
BS
WT
BS
WT
BS
WT
BS
WT
BS
Y35C
BS
C54S/C64S
WT Y35C C64S C54S/C64S C54S
Beads
IB: Claudin-2
Supernatant
IB: Claudin-2
Supernatant
IB: -actin
WT Y35C C64S C54S/C64S C54S
WT
MTSEA-biotin +
WT
-
Beads
IB: Claudin-2
WT
+
WT
-
Supernatant
IB: Claudin-2
MTSEA-biotin
A
B
C

24
The cation pore function is lost in the serine mutants.
The functional consequence of losing the conserved cysteines on the
electrophysiological characteristics of the claudin-2 pore was determined by the
measurement of the transepithelial conductance and NaCl dilution potential in Ussing
chamber. Induction of claudin-2 wild-type expression in MDCK I Tet-off cells increased
the conductance and P
Na+
by 8-fold and 9-fold respectively. By contrast, there was only
very small increase in conductance and P
Na+
in the serine mutants (Figure 2-6A). By
subtracting the average baseline value in the Dox+ conditions from the values in Dox-
conditions, the conductance and P
Na+
attributable to claudin-2 were determined. From
this, we found that the conductance and P
Na+
of each mutant were almost completely
abolished compared to wild-type, whereas there was no statistical significance between
either of the single mutants and the double mutant (Figure 2-6B). The observation that
mutating either one or both conserved cysteines (C54 and C64) impaired conductance
and P
Na+
to a similar extent strongly suggests that the loss of pore function is not an effect
of any individual cysteine but rather is a consequence of disrupting the disulfide bond
between them. We conclude that this disulfide bond is necessary for the formation of a
functional cation pore.

25

Conductance
WT C54S C64S C54S/C64S
0
5
10
15
20
Dox+
Dox-
***
**
Conductance (mS)
Absolute conductance
-5
0
5
10
15
20
WT C54S C64S C54S/C64S
N.S.
Conductance (mS)
Na
+
permeability
WT C54S C64S C54S/C64S
0
10
20
30
40
Dox+
Dox-
***
* **
Permeability (*10
-6
cm/s)
Absolute Na
+
permeability
-10
0
10
20
30
40
WT C54S C64S C54S/C64S
N.S.
Permeability (*10
-6
cm/s)
A
B
Figure 2-6. Characterization of the electrophysiological properties of claudin-2 serine mutants.
(A) The transepithelial conductance (left) and the calculated Na
+
permeability (right) without (Dox+)
or with (Dox-) induction of expression of the indicated claudin-2 constructs. (B) The absolute
conductance (left) and the absolute Na
+
permeability (right) attributable to claudin-2 obtained by
subtracting the average baseline value of the noninduced cells from induced cells. Data points represent
the means of 3-4 filters ± S.E. The p value in (A) was obtained from unpaired Student's t-test between
induced (Dox-) and uninduced (Dox+) state. The p value in (B) was obtained from one-way ANOVA
test with the Bonferroni correction. N.S: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001

26
DISCUSSION
The ECL1 of claudins forms the lining of ion-selective paracellular pores at the
tight junction. The ECL1 signature sequence GLW-[X]
2
-C-[X]
8-10
-C is likely to be
important for correct folding of ECL1. In this study, we find that two conserved cysteines
of ECL1 (C54 and C64 in claudin-2) form an intramolecular disulfide bond that is
necessary for correct pore folding and function.
C54 and C64 in claudin-2 form an intramolecular disulfide bond. A cysteine side-
chain in a protein generally exists in one of three main biochemical states: intermolecular
disulfide-bonded, intramolecular disulfide-bonded, or as a reduced free thiol group. On a
non-reducing SDS-PAGE gel, the vast majority of wild-type claudin-2 migrates as a
monomer. This excludes the idea that C54 or C64 forms an intermolecular disulfide
bond. We do sometimes see a faint dimer band even in wild-type protein (as in Fig. 2),
but it always constitutes an extremely small portion of total claudin-2 protein compared
to the monomer band, and this band does not change in abundance in non-reducing
conditions. We think it could be a result of noncovalent interaction, consistent with the
findings of Van Itallie, who used blue native gels to show that claudin-2 does undergo
spontaneous dimerization mediated by the second transmembrane domain (57). We have
shown previously that there is no free thiol group accessible extracellularly in wild-type
claudin-2 (4). Thus, either C54 and C64 form an intramolecular disulfide bond or, if they
exist as free cysteines, they must both be buried within the protein fold. The following
evidence supports the intramolecular disulfide bond hypothesis. First, mutating either one

27
of the two cysteines induces the remaining cysteine to form a claudin-2 homodimer
linked by an intermolecular disulfide bond. Although it has been reported that C54S and
C64S mutants of claudin-5 do not form a dimer (59), it is possible that this is because the
dimers are reduced under the SDS-PAGE conditions or that C54 and C64 in claudin-5
happen not to be located near the intermolecular interface. Second, mutating either one of
the two extracellular cysteines in claudin-2 makes the other one modifiable by the thiol-
reactive reagent, MTSEA-biotin. It is unlikely that two free cysteines would have a
mutual effect on each other such that mutating either one would make the other
accessible. Collectively, our data support the proposition that the two cysteines of ECL1
normally form an intramolecular disulfide bond. Our inability to reduce the disulfide
bond extracellularly in claudin-2 wild-type indicates either that reducing the disulfide
bond is energetically unfavorable or that the disulfide bond is buried inside the
extracellular fold where it is inaccessible to the reducing agent.
Losing the disulfide bond does not affect claudin-2 trafficking. In general,
mutating conserved amino acids may cause protein misfolding and retention in the
endoplasmic reticulum. In claudin-2, mutating the “GLW” of the signature sequence
causes claudin-2 mislocalization (57). However, mutating the “C-C” of the signature
sequence to serine does not affect claudin-2 trafficking to tight junction. This finding is
consistent with observations by others in claudin-5 (59) and claudin-1 (12), and suggests
that the disulfide bond is not critical for global claudin protein folding.

28
The disulfide bond is necessary for pore function. In our studies, the serine
mutants, C54S, C64S and C54S/C64S, expressed at similar levels to wild-type at tight
junction, yet the transepithelial conductance and P
Na+
attributable to each mutant were
almost abolished. Furthermore, all three mutants lost pore function to the same extent.
This suggests that the impaired pore function is likely caused not by disruption of any
one individual cysteine residue but by disruption of the disulfide bond between them. We
conclude that the role of the extracellular disulfide bond between the two conserved
cysteines is to stabilize ECL1 in its correctly folded conformation and that this is
important for many of the important functions of claudins such as paracellular pore and
barrier formation, and extracellular interactions with other proteins. We believe this role
is generalizable to most or all claudin family members.
C54 and C64 are localized at an intermolecular interface involved in cis-
interaction. Claudins are believed to polymerize within the tight junction, and to be
stabilized by side-by-side interactions between claudin protomers within the same cell
and same tight junction strand (cis-interaction) and by head-to-head interactions between
claudins from the lateral plasma membranes of two adjacent cells (trans-interaction). One
way in which we have been able to identify residues that are located at or close to an
intermolecular interface between claudin proteins is by showing that they form an
intermolecular disulfide bond when they are mutated to cysteine, for example D65 in
claudin-2 (5). All four of our C54 and C64 single mutants were shown to form claudin-2
homodimers linked by intermolecular disulfide bonds, suggesting that C54 and C64 are

29
located at or close to an intermolecular interface. In the case of C54A and C64A, the
dimerization presumably occurs intracellularly since these mutants did not localize at the
tight junction. Since therefore there is no possibility of trans-interaction, these results
strongly suggest that the two conserved cysteines are localized at the cis-interaction
interface. The cysteines at the cytoplasmic end of the second transmembrane domain of
claudin-2 have also been shown to be in proximity to an intermolecular interface for cis-
interaction (57). These two findings suggest that claudin-2 can homodimerize within the
cell, presumably as early as in the endoplasmic reticulum, via both the transmembrane
and extracellular domains. Whether C54 and C64 could in addition be close to a trans-
interaction interface requires further study.
Mutation of the extracellular conserved cysteines leads to familial
hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC). FHHNC is a rare
autosomal recessive renal tubule disorder characterized by renal magnesium and calcium
wasting, and nephrocalcinosis. It is the result of a defect in divalent cations reabsorption
in the thick ascending limb of Henle, due to claudin-16 (46) or claudin-19 mutations (31).
One such mutation, claudin-16 C120R, was reported in a patient with FHHNC (48). C120
is predicted to be an extracellular cysteine in claudin-16 and is homologous to C54 in
claudin-2. The finding that mutation of this extracellular cysteine can cause clinical
disease supports our conclusion that the conserved cysteines of ECL1 are critical for its
pore function.

30
In conclusion, the extracellular conserved cysteines form an intramolecular
disulfide bond that is necessary for pore function in claudin-2. This finding, along with
observations about the critical role of these cysteines in claudin-1 (10, 12), claudin-5
(59), and claudin-16 (48), suggests that the correct folding and hence proper function of
ECL1 require the two conserved cysteines to form an intramolecular disulfide bond. We
speculate that the role of these two cysteines is to stabilize an extracellular fold that is
common to all claudins.

31
CHAPTER 3: CONSERVED AROMATIC RESIDUE CONFERS
CATION SELECTIVITY IN CLAUDIN-2 AND CLAUDIN-10B
ABSTRACT
In tight junctions, both claudin-2 and claudin-10b form paracellular cation-
selective pores by the interaction of the first extracellular loop (ECL1) with permeating
ions. We hypothesized that a highly conserved aromatic residue near the pore selectivity
filter of claudins contributes to cation selectivity by cation-pi interaction with the
permeating cation. To test this, we generated MDCK I Tet-off cells stably transduced
with claudin-2 Y67 mutants. Y67L showed reduced cation selectivity compared to wild-
type claudin-2 due to a decrease in Na
+
permeability, without affecting the Cl
-

permeability. Y67A enlarged the pore size and further decreased the charge selectivity
due to an increase in Cl
-
permeability. Y67F restored the Na
+
permeability, Cl
-

permeability, and pore-size back to wild-type. The accessibility of Y67C to
methanethiosulfonate modification indicated that its side chain faces the lumen of the
pore. In claudin-10b, F66L reduced cation selectivity and F66A lost the pore
conductance. We conclude that the conserved aromatic residue near the cation pore
domain of claudins contributes to cation selectivity by a dual role of cation-pi interaction
and a luminal steric blocking effect. Our findings provide new insight into how ion
selectivity is achieved in the paracellular pore.

32
INTRODUCTION
Epithelial cells are connected via multiple junctional complexes. The tight
junction separates the apical and basolateral membrane domains and acts as the
paracellular barrier, while remaining selectively permeable to ions and water. In tight
junctions, the first extracellular loop (ECL1) of claudin forms the paracellular pore or
barrier (6). Both claudin-2 and claudin-10b can form paracellular cation pores with
P
Na+
/P
Cl-
of 6 to 8 (45, 58, 63). The pore diameter of claudin-2 is estimated to be 6.5-7.5
Å (56, 63). The primary determinant of claudin-2 ion charge selectivity is an aspartate
residue in ECL1 (D65) (26, 63). When all three negatively charged residues in the
claudin-2 ECL1, including D65, were mutated to neutral amino acids, the pore became
less cation-selective. However, it remained four times more selective to Na
+
than to Cl
-
(63). This observation led us to postulate that other mechanisms may also play a role in
cation selectivity, such as cation interaction with polar residues (e.g. carbonyl oxygen, as
the case in the KcsA potassium channel (13)), or cation-pi interactions. The latter
possibility prompted us to search for a conserved aromatic residue near D65 and I66,
where the cation-selective filter is located (5, 63). We found position 67 of claudin-2 and
position 66 of claudin-10b to have an aromatic residue that is highly conserved in all of
the classic claudins (tyrosine in claudin-2 and phenylalanine in claudin-10b). The goal of
this study was to assess the role of this aromatic residue in cation pore-forming claudins.
We hypothesized that Y67 (claudin-2) or F66 (claudin-10b) interacts with
permeating cations through cation-pi interaction. Cation-pi interaction is defined as the

33
interaction between positively charged molecules and negatively charged π electrons on
the benzene ring of the aromatic amino acid side chain. Cation-pi interaction has been
identified in the nicotinic receptor ligand binding site (67), as well as in the binding site
for tetraethylammonium (TEA) in potassium channel (21). To test whether Y67 and F66
interact with the permeating cations through cation-pi interaction, we mutated this
aromatic residue to leucine, a bulky and hydrophobic residue without the benzene ring.
By eliminating the cation-pi interaction, both claudin-2 Y67L and claudin-10b F66L were
predicted to be less cation-selective than its respective wild-type protein.
Aromatic residue may also have a role via its steric effect. Its bulky benzene
group could have a mechanical effect to modulate protein conformation and hence
function. In the ATP-sensitive K channel, a pore-lining phenylalanine gates the channel
by steric hindrance (44), and a phenylalanine residue couples activation and inactivation
of the KcsA channel (9). To test whether the conserved aromatic residue exerts a steric
effect, we substituted Y67 (claudin-2) or F66 (claudin-10b) with an alanine, a smaller
hydrophobic residue.
Our findings suggest that the conserved aromatic residue confers cation selectivity
in cation pore-forming claudins by interacting with the permeating cation both via cation-
pi interaction and by restricting the pore size via its steric effect.
MATERIALS AND METHODS
Generation and screening of MDCK I Tet-off claudin-2 and claudin-10b cell
lines. MDCK I Tet-off cells expressing wild-type claudin-2, wild-type claudin-10b,

34
claudin-2 mutants (Y67L, Y67A, Y67C, D65N/Y67L, Y67F), and claudin-10b mutants
(F66L, F66A) were generated by methods described previously (62). In short, the
mutants of mouse claudin-2 and human claudin-10b were generated by site-directed
mutagenesis on the template plasmid, pRevTREP-mouse-claudin2-wt and pRevTREP-
human-claudin10b-wt respectively, using the QuikChange Kit (Stratagene). The plasmids
were lipofected into the viral packaging cell line, PT67. Viral particles were collected
from the growth medium of PT67 cells and used to transduce MDCK I Tet-off cells.
After 7-10 days in a 0.3 mg/ml hygromycin selective medium, independent clones of
MDCK I Tet-off cell lines with transduced constructs were selected using cloning
cylinders. To induce protein expression, doxycycline was omitted from the culture
medium; otherwise 50 ng/mL doxycycline was included to suppress the protein
expression.
Immunoblotting. Protein expression was tested by SDS-PAGE and
immunoblotting. Confluent cells grown on tissue culture dishes were mechanically lysed
by passing through a 25-gauge needle 10 times in sucrose-histidine lysis buffer
containing 0.25M sucrose, 30mM histidine, 1mM EDTA (pH 8), and protease inhibitor
(Complete Mini, Roche Diagnostics). Cell lysates were loaded in reducing SDS-PAGE
buffer (1% (v/v) 2-mercaptoethanol added) and heated at 75˚C for 10 minutes. 20µg of
protein samples were loaded on 12% polyacrylamide gel, transferred to a PVDF
membrane, blotted with 1:500 mouse anti-claudin-2 antibody (Life Technologies) or
1:500 rabbit anti-claudin-10b antibody (Life Technologies), and then appropriate

35
horseradish peroxidase-conjugated secondary antibodies (GE), detected with the
chemiluminescent method (Pierce), and imaged by an ImageQuant LAS-4000 (GE
Healthcare).
Immunofluorescent staining. The cells were plated at a density of 10
5
cells/1.16
cm
2
on 12-well Transwell plates and grown for seven days. The cells were washed in ice-
cool phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) at 4˚C for
15 minutes, permeabilized and blocked in a permeation buffer (0.3% Triton X-100, 1%
BSA, 5% goat serum in PBS) for one hour. The filters were incubated in primary
antibodies (1:500 mouse anti-claudin-2 and 1:500 rabbit anti-ZO-1; or 1:500 rabbit anti-
claudin-10b and 1:500 mouse anti-ZO-1) for two hours at room temperature, washed in
PBS, incubated in secondary antibodies (for claudin-2 staining: Alexa Fluor 488-
conjugated anti-mouse IgG and Alexa Fluor 555-conjugated anti-rabbit IgG, both 1:1000;
for claudin-10b staining: Alexa Fluor 555-conjugated anti-mouse IgG and Alexa Fluor
488-conjugated anti-rabbit IgG, both 1:1000) for one hour, washed in PBS, and mounted
in ProLong anti-fade mounting medium. All the reagents were from Life Technologies.
Slides were imaged by a Leica TCS SP2 multi-photon confocal microscope.
Electrophysiological studies in Ussing chamber. Cells were plated at a density of
10
5
cells/1.16cm
2
on Snapwell filters (Corning) and cultured for seven days in the
presence (Dox+) or absence (Dox-) of 50 ng/ml doxycycline. The Ussing chamber setup
and liquid junction potential correction method was employed as previously described
(62). The conductance and permeability attributable to claudin-2 pore was calculated by

36
subtracting the average value of the uninduced (Dox+) state from the values of the
induced (Dox-) state. The standard Ringer solution used at baseline contained (in mM):
NaCl 150, CaCl
2
2, MgCl
2
1, glucose 10, Tris-HEPES 10, pH 7.4. To measure Na
+

permeability, the solution in the basolateral chamber was changed to 75mM NaCl Ringer
solution (osmolarity adjusted with mannitol). To measure alkali metal biionic potential,
the basolateral medium was changed to 150mM alkali metal chloride salt. To measure
organic cation permeability, the basolateral medium was changed to the solution
containing 75mM organic cation chloride salt and 75mM NaCl. The organic cations
included methylamine (MA), ethylamine (EA), tetramethylammonium (TMA),
tetraethylammonium (TEA). The ion permeability β=P
Cl
-
/P
Na
+
was calculated from the
Goldman-Hodgkin-Katz voltage equation. The absolute Na
+
permeability was estimated
by the method devised by Kimizuka and Koketsu (29). The alkali metal permeability was
calculated from γ=P
M
/P
Na
, where γ was estimated as (1):
! = (1+")•e
v/
RT
F
!" (1)
The organic cation permeability was calculated from the following equation:
! ="(1+#)•e
V/
RT
F
!"#!1 (2)
Here, α represents the activity ratio of NaCl in the apical compartment over the
basolateral compartment. The activity of NaCl at 150mM is 0.752, and 0.797 at 75mM,
resulting in α =(150*0.752)/(75*0.797) = 1.89.

37
The pore size was estimated by the method previously described (63). In short, the
claudin-2 pore was assumed to be a cylinder of diameter, D, across which cations of
diameter, d, diffused. According to the Renkin equation (3):
P=
A
d
1!
d
D
"
#
$
%
&
'
2
(3),
The square root of the relative permeability of MA, EA, and TEA to Na
+
is linearly
related to the cation diameter. D was estimated from the x-intercept of the best-fit line, as
determined by linear regression.
Substituted cysteine modification assay. To test the effect of the cysteine-
modifying reagents on pore function, [2-(trimethylammonium) ethyl]
methanthiosulfonate (MTSET) and benzyl methanethiosulfonate (MTS-Bn) were added
to the cells, and the changes of pore conductance and NaCl dilution potentials were
measured. The working concentration of MTSET and MTS-Bn was 1mM. To avoid
hydrolysis, the reagent was freshly dissolved as 100-fold concentrated stock solution
immediately before starting the experiment. The stock solution was added simultaneously
to the medium of the apical and basolateral chambers and rapidly mixed by gas lifts. The
change in conductance was calculated by percentage decrease of conductance from the
pre-treatment state to the five minutes post-treatment state. The NaCl dilution potential
was measured before and after the treatment.
Cysteine-specific surface biotinylation. To test the accessibility of the substituted
cysteine, the cysteine-specific surface biotinylation was performed. Cells were plated at a

38
density of 5×10
5
cells/well on a 6-well plate and grown for six days. Cells were treated
with 0.5ml/well freshly dissolved 2-((biotinoyl)amino)ethyl methanethiosulfonate
(MTSEA-biotin) in PBS/CM at a concentration of 0.5mg/ml. The plate was incubated at
room temperature for 10 minutes, washed three times with ice-cold PBS, and the cells
were harvested in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% (w/v) SDS,
0.5% (w/v) deoxycholic acid, 1% (v/v) NP-40). The cell lysates were centrifuged at
16,000 g for 15 minutes. The supernatant was added to a 40µl slurry of streptavidin-
coated beads and rotated at 4˚C for two hours. The beads were then pelleted and the
supernatant was saved for analysis. The beads were washed three times in TBS (50mM
Tris-HCl and 150mM NaCl), added to 20µl 2x reducing SDS-PAGE loading buffer, and
heated at 75˚C for 10 minutes with occasional agitation. Both the bead (biotinylated
protein fraction) and supernatant (non-biotinylated fraction) samples were then subjected
to immunoblotting as described above.
Statistics. The data are presented as mean ± standard error. Statistical significance
was determined using unpaired two-tailed Student’s t-test or one-way ANOVA test. The
P value of multiple comparisons was corrected using the Bonferroni correction. P< 0.05
was considered to be statistically significant.

39
RESULTS
Stable transduction of claudin-2 and claudin-10b mutants in MDCK I Tet-off cells.
To test the role of the aromatic residue near the pore selective filter, claudin-2
constructs (Y67L, Y67A, Y67C, Y67F, and D65N/Y67L) and claudin-10b constructs
(wild-type, F66L, F66A) were transduced into MDCK I Tet-off cells using retroviral
transduction, and stably transduced clones were selected. Inducible protein expression
was verified by immunoblotting, which shows a characteristic band of claudin-2
monomer (Figure 3-1A) and a band of claudin-10b monomer (Figure 3-1C) at
approximately 20kD in the absence of doxycycline. There were also multiple bands less
than 20kD in the claudin-2 blot, which are not seen in the claudin-2 blot of mouse kidney
lysates (data not shown). They are therefore probably proteolysis products, which we
often see in overexpressing proteins in cells. Immunofluorescent staining of claudins and
ZO-1 shows that all the claudin-2 constructs (Figure 3-1B) and claudin-10b constructs
(Figure 3-1D) were localized at the tight junction.

40

Figure 3-1. Characterization of MDCK I Tet-Off cell lines stably transduced with claudin-2 and
claudin-10b constructs.
(A) Immunoblot of cell lysates from stable clones of claudin-2 constructs (WT, D65N, I66C, Y67L,
Y67A, Y67C, Y67F, and D65N/Y67L) and (C) claudin-10b constructs (WT, F66A, and F66L), grown
in the presence (+) or absence (-) of doxycycline (Dox). Cell lysates were subjected to reducing SDS-
PAGE and immunoblotted with anti-claudin-2 antibody or anti-claudin-10b antibody. The image was
exposed for 15 minutes. Inducible expression of claudin-2 monomer and claudin-10b monomer was
seen at approximately 20kD all constructs. Localization of (B) claudin-2 and (D) claudin-10b constructs
shown by immunofluorescent staining of ZO-1 (red) and claudin-2 or claudin-10b (green). All mutants
co-localized with ZO-1 at the tight junction.

41
In claudin-2, leucine substitution leads to partial loss of cation selectivity without
affecting the pore size.
To test whether Y67 contributes to cation selectivity by cation-pi interaction, we
mutated this position to leucine, a bulky and hydrophobic residue without the benzene
ring. Y67L was predicted to be less cation-selective than wild-type. Consistent with this,
the P
Na
+
/P
Cl
-
ratio of claudin-2 Y67L was 5.4±0.3, which was significantly smaller than
wild-type claudin-2 (12.5±1.8) (Figure 3-2A). If both D65 and Y67 function as sites that
independently confer cation selectivity, the P
Na
+
/P
Cl
-
of D65N/Y67L should be further
reduced from the single mutants. The P
Na
+
/P
Cl
-
of D65N/Y67L was 2.9±0.2, a significant
decrease from D65N (8.1±0.5). In addition, it was approximately half the P
Na
+
/P
Cl
-
of
Y67L, although the difference did not reach a level of statistical significance.
Compared to wild-type, the partial decrease in the cation selectivity of Y67L was
due to a significant decrease in Na
+
permeability (Figure 3-2B) without affecting the
Cl
-
permeability (Figure 3-2C). The P
Na
+
of D65N/Y67L was less than Y67L, and the P
Cl
-
was higher than Y67L. Neither, however, reached a level of statistical significance. The
relative permeability of Y67L to alkali metal cations (Figure 3-2D, orange line) was
identical to wild-type. As shown previously (63), the relative permeability to Li
+

(P
Li
+
/P
Na
+
) of D65N (Figure 3-2D, blue line) was less than wild-type, indicating a loss of
a strong intrapore binding site for dehydrated cations. In contrast, the P
Li
+
/P
Na
+
of Y67L
was no different than wild-type. The relative permeability of D65N/Y67L to Li
+
was
similar to D65N. The phenotype of the double mutation appears to be additive of the

42
effect of the two single mutants, suggesting that D65 and Y67 are two distinct sites that
independently confer cation selectivity. The pore diameter (in Å) of Y67L and
D65N/Y67L was estimated to be 6.7±0.2 and 6.1±0.5 (Figure 3-2E) respectively, neither
of which was significantly different from wild-type (6.6±0.2).
In summary, the leucine substitution in the aromatic residue of claudin-2
decreased the cation selectivity due to a decrease in Na
+
permeability without affecting
Cl
-
permeability or the pore size.

43

Estimation of pore size
345678
0.0
0.5
1.0
1.5
MA
+
EA
+
TMA
+
***
Cldn2 WT
Y67F
D65N
Y67L
D65NY67L
Y67A
Ionic diameter (Å)
Sqaure root of P
M
+
/P
Na
+
Na
+
permeability
WT
D65N
Y67L
D65NY67L
Y67A
Y67F
0
10
20
30
***
**
N.S.
N.S.
Permeability (*10
-6
cm/s)
Relative permeability to Na
+

1234567
0.0
0.5
1.0
1.5
2.0
Li
+
Na
+
Rb
+
Cs
+ K
+
MA
+
TMA
+
TEA
+
EA
+
Cldn2 WT
Y67F
D65N
Y67L
D65NY67L
Y67A
Ionic diameter (Å)
P
M
+
/P
Na
+
Cation selectivity
WT
D65N
Y67L
D65NY67L
Y67A
Y67F
0
5
10
15
20
25
***
***
* ***
***
N.S.
***
N.S.
P
Na
+
/P
Cl
-
A
E D
BC
Cl- permeability
WT
D65N
Y67L
D65NY67L
Y67A
Y67F
0
2
4
6
8
N.S.
N.S.
N.S.
N.S.
***
Permeability (*10
-6
cm/s)
Figure 3-2. Characterization of the electrophysiological properties of claudin-2 constructs.
MDCK I Tet-off cells transduced with claudin-2 constructs (WT, D65N, Y67L, D65N/Y67L, Y67A,
and Y67F) were plated at a density of 10
5
cells/1.16cm
2
and grown for seven days before mounting in
Ussing chamber. (A) The permeability ratio was calculated as P
Na
+
/P
Cl
-
, where P
Na+
and P
Cl-

were
calculated from NaCl dilution potentials and subtracting the average baseline permeability of uninduced
(Dox+) cells from that of the induced (Dox-) cells. (B) Na
+
permeability and (C) Cl
-
permeability of
claudin-2 WT, Y67F, D65N, Y67L, D65N/Y67L, and Y67A. (D) The permeability of claudin-2
constructs (WT, Y67F, D65N, Y67L, D65N/Y67L, and Y67A) to alkali metal cations and organic
cations relative to their Na
+
permeability were plotted against the ionic diameters. (E) The relationship
between the square roots of the relative permeability of methylamine (MA), ethylamine (EA), and
tetramethylammonium (TMA) and ionic diameters were fitted by linear regression, and the pore
diameter was estimated as the x-intercept of the best-fit line. Data points represent the means of 3-9
filters ± SE. *P<0.05, ** P <0.01, *** P <0.001. P values were obtained from one-way ANOVA test
with the Bonferroni correction.

44
In claudin-2, alanine substitution abolishes cation selectivity and enlarges pore size
To test whether the aromatic group exerts a steric effect, we substituted an amino
acid with a side chain that was as small as possible. Glycine is the smallest amino acid,
but it would introduce flexibility to the peptide backbone. Instead, alanine was selected
because it has the second smallest side chain, and because its substitution is usually well
tolerated and only infrequently causes protein misfolding.
Mutation of Y67 to alanine eliminates both the benzene ring and the bulky side
chain. Thus, Y67A was compared to Y67L in order to specifically pinpoint the role of
steric effects from the bulky side-chain. The P
Na
+
/P
Cl
-
of Y67A was 1.4±0.1, smaller than
the P
Na
+
/P
Cl
-
of Y67L (Figure 3-2A). The cation selectivity of Y67A approached the ratio
of mobilities of these ions in free solution (P
Na
+
/P
Cl
-
=0.7) (30). Thus, Y67A almost
completely abolished the cation selectivity of claudin-2. Compared to Y67L and
D65N/Y67L, the decrease in the cation selectivity in Y67A was due to a significant
increase of Cl
-
permeability (Figure 3-2C) without further affecting Na
+
permeability
(Figure 3-2B). In Y67A, the relative permeability of large alkali metal and organic
cations (Figure 3-2D, red line) was significantly increased from wild-type. The estimated
pore size of Y67A was 7.6±0.1Å (Figure 3-2E), which was significantly larger than that
of wild-type, D65N, Y67L, and D65N/Y67L.
In summary, alanine substitution almost completely abolished the cation
selectivity of claudin-2 due to an increase in Cl
-
permeability without affecting Na
+

45
permeability. The pore size of Y67A was significantly enlarged from Y67L and wild-
type, suggesting that Y67 restricted the pore size by a steric effect.
In claudin-2, substitution of another aromatic residue at position 67 partially
restores cation selectivity and pore size.
If a bulky aromatic ring at position 67 confers cation selectivity, substitution of
phenylalanine at this position should have a similar function. To test this, we made the
claudin-2 mutation, Y67F. Y67F partially restored cation selectivity as evidenced by a
P
Na
+
/P
Cl
-
of 5.9±0.4, which was significantly greater than Y67A (Figure 3-2A). The P
Na
+

and P
Cl
-
of Y67F were not statistically significantly different from wild-type (Figure 3-2B
and 3-2C), and the relative cation permeability curve (Figure 3-2D) and the pore size
(Figure 3-2E) were almost identical to wild-type.
In claudin-2, the side chain of residue 67 is accessible from the pore lumen.
There are two possible side chain conformations that Y67 could adopt that would
restrict the pore size. The side chain could point directly into the pore lumen.
Alternatively, the side chain could be buried inside the protein fold and sterically push
the pore lining residues into the pore lumen. To determine the conformation of the Y67
side chain relative to the pore, we generated a Y67C mutant and assessed the accessibility
of the substituted cysteine to membrane-impermeable methanethiosulfonate (MTS)
reagents. As positive controls, we used cysteine mutants of a known pore-lining residue
in claudin-2, I66, and of a residue known to face the outside of the pore, Y35 (5). If the

46
side chain at position 67 points towards the pore lumen, Y67C will be accessible to
extracellularly applied MTS reagents.
Y67C had lower P
Na
+
/P
Cl
-
than wild-type, consistently suggesting cation-pi
interaction has an important role in cation selectivity (Figure 3-3A). Y67C had higher
relative permeability of cations larger than Na
+
(Figure 3-3B), and the estimated pore size
of Y67C (7.1 ± 0.4 Å) was significantly increased compared to wild-type (Figure 3-3C).
Next, we extracellularly applied MTSEA-biotin to probe the accessibility of the
substituted cysteine. Y67C was biotinylatable (Figure 3-3D), suggesting that the amino
acid side chain of Y67 was not folded within the protein. The biotinylated fraction of
Y67C was similar to Y35C in abundance, and was much greater than I66C, suggesting
that Y67C was more accessible than I66C, likely reflecting the enlarged pore size.

47

Relative permeability of cations to Na
+

1234567
0.0
0.5
1.0
1.5
2.0
Li
+
Na
+
Rb
+
Cs
+
K
+
MA
+
TMA
+
TEA
+
EA
+
Cldn2 WT
Y67C
Ionic diameter (Å)
P
M
+
/P
Na
+
Estimation of pore size
345678
0.0
0.5
1.0
1.5
Cldn2 WT
MA
+
EA
+
TMA
+
Y67C
***
Ionic diameter (Å)
Sqaure root of P
M
+
/P
Na
+
A
CD
Cation selectivity
WT
Y67C
0
5
10
15
P
Na
+
/P
Cl
-
***
B
Figure 3-3. Characterization of the structural-functional properties of claudin-2 Y67C.
(A) Cation selectivity of Y67C (B) The permeability of claudin-2 constructs (WT and Y67C) to alkali
metal cations and organic cations relative to their Na
+
permeability were plotted against the ionic
diameters. (C) The square roots of the relative permeability of methylamine (MA), ethylamine (EA),
tetramethylammonium (TMA) were fitted by linear regression and the pore diameter was estimated as
the x-intercept. (D) Cells expressing claudin-2 Y35C, Y67C, I66C, and WT were treated with MTSEA-
biotin, followed by streptavidin precipitation. The bead fraction and the supernatant fraction were
subjected to SDS-PAGE and blotted with anti-claudin-2 antibody. The upper blot shows the
biotinylated claudin-2 on the beads. The lower blot shows the non-biotinylated claudin-2 in the
supernatant as the loading control. Data points represent the means of 3 filters ± SE. *P<0.05, ** P
<0.01, *** P <0.001. P values were obtained from one-way ANOVA test with the Bonferroni
correction.

48
If the side chain of Y67 faces the pore lumen, MTS reagents might be expected to block
the pore. However, we found that neither MTSET (Figure 3-4A) nor MTSEA-biotin (data
not shown) altered the conductance of Y67C or ion selectivity (data not shown).
Furthermore, when we attempted to restore a benzene group to Y67C by adding benzyl
MTS (MTS-Bn), the MTS-Bn treatment neither changed the conductance of Y67C, nor
the Na
+
or Cl
-
permeability (Figure 3-4B), suggesting that the MTS reagents may not
mimic the actual Y67 side chain confirmation in the wild-type protein.
In summary, like alanine, cysteine substitution in Y67 enlarged the pore size.
Y67C was accessible from the aqueous environment, but MTS reagents were unable to
block the pore conductance.

MTSET conductance inhibition asay
I66C
Y67C
-25%
-20%
-15%
-10%
-5%
0%
***
Change of conductance in percentage
MTS-Bn Assay
Conductance
Na
+
Cl
-
0
2
4
6
8
0
5
10
15
Before MTS-Bn
After MTS-Bn
N.S. N.S.
N.S.
Conductance (mS)
Permeability (*10
-6
cm/s)
AB
Figure 3-4. Conductance inhibition assay by MTSET and MTS-Bn.
(A) MTSET inhibition assay in Ussing chamber. The change of conductance was calculated as the
percentage change in the conductance at 5-minute after addition of MTSET compared to pre-treatment.
(B) MTS-Bn inhibition assay in Ussing chamber. The conductance and NaCl dilution potential were
measured before adding 1mM MTS-Bn and measured 5 minutes after the treatment. The Na
+
and Cl
-

permeability were calculated from the conductance and dilution potential.

49
F66 is critical for the function of claudin-10b.
To determine if the findings of Y67 in claudin-2 are generalizable to other cation
pore-forming claudins, we generated MDCK I Tet-off cells expressing claudin-10b wild-
type, F66L, and F66A. F66 is the aromatic residue that is homologous to Y67 in claudin-
2.
The pore properties of wild-type claudin-10b were consistent with previous
findings (45, 58) (Figure 3-5). In
brief, claudin-10b increased the
transepithelial conductance by
approximately 6.5 fold. It was
four times more permeable to Na
+

and the order of relative
permeability to alkali metal
cations was that of Eisenman
sequence VIII.

Cation selectivity
Claudin-10b WT
0
1
2
3
4
5
P
Na
+
/P
Cl
-
AB
CD
Conductance
Dox+
Dox-
0
5
10
15
Conductance (mS)
Relative permeability
1234567
0.0
0.5
1.0
1.5
Li
+
Na
+
Rb
+
Cs
+
K
+
MA
+
TMA
+
TEA
+
EA
+
Ionic diameter (Å)
P
M
+
/P
Na
+
Permeability
0
5
10
15
20
Na
+
Cl
-
Permeability (*10
-6
cm/s)
Figure 3-5. Characterization of the electrophysiological properties of claudin-10b wild-type (WT).
MDCK I Tet-off cells transduced with claudin-10b WT were plated at a density of 10
5
cells/1.16cm
2
and
grown for seven days before mounting in Ussing chamber. (A) Change of conductance from the
uninduced state (Dox+) to the induced state (Dox-) (B) Cation selectivity presented as P
Na
+
/P
Cl
-
, where
P
Na+
and P
Cl-

were calculated from NaCl dilution potentials and subtracting the average baseline
permeability of uninduced (Dox+) cells from that of the induced (Dox-) cells. (C) Na
+
permeability and
Cl
-
permeability. (D) The relative permeability of alkali metal cations and organic cations relative to
their Na
+
permeability were plotted against the ionic diameters. Data points represent the means of 3
filters ± S.E.

50
Similar to claudin-2 Y67L, the P
Na
+
/P
Cl
-
ratio of claudin-10b F66L was 1.3±0.3
(Figure 3-6B), which was significantly less than that of wild-type claudin-10b (4.2±0.4).
The decrease of cation selectivity of F66L was due to reduced Na
+
permeability without
changing Cl
-
permeability (Figure 3-6C). This suggests that the role of the aromatic
residue at this site is generalizable to other cation pore-forming claudins.

Interestingly, claudin-10b F66A did not increase the conductance of MDCK I
cells at all (1.61±0.28 mS in Dox+ and 1.40±0.11 mS in Dox-), suggesting that it was not
functional (Figure 3-6A). Thus, we were unable to compare the effect of F66A on the
pore size and charge selectivity of claudin-10b to the effect of Y67A on claudin-2.
Conductance
WT
F66A
F66L
0
5
10
15
Dox+
Dox-
*** ***
*
Conductance (mS)
Permeability
WT
F66L
0
5
10
15
20
Na
+
permeability
Cl
-
permeability
*
N.S.
Permeability (*10
-6
cm/s)
Cation selectivity
WT
F66L
0
1
2
3
4
5 ***
P
Na
+
/P
Cl
-
AB C
Figure 3-6. Characterization of the electrophysiological properties of claudin-10b constructs.
MDCK I Tet-off cells transduced with claudin-10b constructs (WT, F66L, and F66A) were plated at a
density of 10
5
cells/1.16cm
2
and grown for seven days before mounting in Ussing chamber. (A) Change
of conductance from the uninduced state (Dox+) to the induced state (Dox-). (B) The permeability ratio
was calculated as P
Na
+
/P
Cl
-
, where P
Na+
and P
Cl-

were calculated from NaCl dilution potentials and
subtracting the average baseline permeability of the uninduced (Dox+) cells from that of the induced
(Dox-) cells. (C) Na
+
permeability and Cl
-
permeability of claudin-10b WT and F66L. Data points
represent the means of 3 filters ± SE. *P<0.05, ** P <0.01, *** P <0.001. P values were obtained from
unpaired Student’s t-test.

51
DISCUSSION
Claudin-2 and claudin-10b are cation-selective pores at the tight junction. In
claudin-2, mutating all three negatively charged amino acids in the pore-forming first
extracellular domain makes the pore become less cation-selective. However, the pore still
remains four times more permeable to Na
+
than to Cl
-
, suggesting that other non-charged
amino acids may also contribute to the cation selectivity. Y67 and F66 are conserved
aromatic residues in claudin-2 and claudin-10b, respectively, that are located near the
pore selectivity filter. We initially hypothesized that Y67 (F66) contributes to cation
selectivity by side chain cation-pi interaction with the permeating cation. We found that
this aromatic residue in cation claudin pores was required for cation selectivity because
of a dual role: facilitating cation permeation by cation-pi interaction and preventing anion
permeation by a luminal steric effect.
In claudin-2, Y67 provides the minor interaction site for the permeating cation by
cation-pi interaction. Na
+
is hydrated in solution. The hydration enthalpy for Na
+
is -405
kJ/mol (61). In wild-type claudin-2, cations permeate through the pore in a partially
dehydrated form (63). The majority of the energy for dehydration comes from the
electrostatic interaction of the cations with D65 within the pore (63). In addition to D65,
Y67 seems to play a role. Y67L decreases Na
+
permeability without a change in Cl
-

permeability, alkali metal cation permeability pattern, or the pore size. This suggests that
Y67L loses the ability to facilitate Na
+
permeation rather than alters the pore
conformation. The most likely explanation is that Y67 facilitates Na
+
permeation by

52
cation-pi interaction. Cation-pi interaction could provide 16-125 kj/mol of binding energy
(19), and it is generally weaker than electrostatic interaction. The quantitative
measurement for the binding energy of Na
+
to D65 and Y67 is not available because the
stoichiometry of claudin-2 pore is not known. However, D65N was much less permeable
than wild-type for the heavily hydrated cation, Li
+
(63), while Y67L did not significantly
decrease the relative permeability of Li
+
. This relationship suggests that D65 provides the
major portion of cation permeation energy cost, and Y67 contributes a minor portion of
that, in agreement with the magnitude of strength of electrostatic interaction and cation-pi
interaction. Furthermore, the double mutant D65N/Y67L was less cation selective than
D65N, reflecting the additive cation selective effect of Y67. Meanwhile, the P
Li
+
/P
Na
+

ratio of D65N/Y67L was less than Y67L, reflecting the loss of the strong intrapore ion-
binding site: D65. This suggests that D65 and Y67 are two distinct sites that
independently confer cation selectivity.
In claudin-2, Y67 restricts the pore size by steric effect to prevent Cl
-
permeation.
The claudin-2 pore is 6.5–7.5 Å in diameter, and the hydrated diameter of Na
+
and Cl
-
is
estimated to be 9.4 Å and 7.8 Å respectively (28). Because Na
+
can be partially
dehydrated within the pore, and hence has a smaller hydrated diameter than Cl
-
, Na
+
is
more permeable than Cl
-
in wild-type claudin-2.

In Y67A, the pore is enlarged by 0.8–
1.2Å, which allows ions to diffuse without dehydration. Because Cl
-
is more mobile than
Na
+
in free diffusion, Y67A increases Cl
-
permeability disproportionately to Na
+

permeability. A similar pore enlarging effect is seen in Y67C, precluding the explanation

53
that the pore enlarging effect is an artifact of the introduced amino acid. Comparing the
substitution of alanine with that of leucine at this site, Y67A lacks the bulky side chain. A
bulky side chain could potentially exert a steric effect on channel gating (44) and
coupling (9). However, the most likely explanation for our results is that a bulky side
chain at position 67 restricts the pore size by a steric effect.
In claudin-2, the side chain of Y67 likely points towards the pore lumen. There are
two possible side chain conformations for Y67 that could restrict the pore size. The side
chain could directly protrude into the pore lumen. Less directly, the side chain could fold
inside the protein and push the pore-lining residues into the pore lumen. Y67C is
structurally accessible to MTSEA-biotin, excluding the possibility that the side chain is
folded within. Whether the side chain points towards the lumen, as is the case with I66,
or on the outside surface of the protein, as is the case with Y35, is debatable. After
MTSEA-biotin exposure, the biotinylated fraction of Y67C is much greater than that of
I66C and similar to Y35C. This may be the result of Y67C being on the outside of the
protein. However, this interpretation does not explain why the Y67 mutants have
dramatically altered the pore properties. Moreover, Y67 is embedded in the middle of a
series of consecutive pore-lining residues: D65 (63), I66 (5), and S68 (details in Ch.4). It
is unlikely that Y67C is facing outside while its two neighboring residues are lining the
pore. We therefore conclude that the Y67 side chain most likely faces to the pore lumen,
and that the high biotinylation fraction is due to the enlarged pore size and hence
increased accessibility to MTSEA-biotin.

54
In claudin-10b, F66 is critical for the pore function. Claudin-10b is also a cation
pore. In the mutagenesis study of F66, the F66L mutant reduced the cation selectivity as
Y67L did in claudin-2. Interestingly, the F66A mutant did not enlarge the pore size as
Y67A did in claudin-2, but instead disrupted the cation pore function of claudin-10b.
This indicates that F66 is a critical residue for the function of claudin-10b.
The dual role of the aromatic residue in ion-selectivity mechanism of claudin
pores. Figure 3-7 shows a homology alignment of part of the first extracellular domain of
the major pore-forming claudins and their charge selectivity. All claudins have two
conserved extracellular cysteines separated by 8-10 residues. Counting from the second
extracellular cysteine, all of the pore claudins have a major charge selectivity site (D, E,
R, or K) located at the +1 and/or +2 position, and 1-2 aromatic amino acid residues
located within the +2 to +4 positions. In cation-selective pore claudins, the role of the
aromatic residue(s) is to enhance the cation selectivity: first, by facilitating Na
+

permeation by cation-pi interaction; second, by preventing hydrated Cl
-
permeation by a
steric effect. In anion-selective pore claudins, we speculate that the presence of a
positively charged binding site overrides the effect of the pi electrons and facilitates
stabilization of a dehydrated Cl
-
ion in the pore, and hence Cl
-
permeation. Concurrently,
the steric effect prevents the hydrated Na
+
ions from permeating.

55

In conclusion, we demonstrate that the conserved aromatic residue located 1-2
residues downstream of the major charge selective site has a dual role for cation
selectivity. It facilitates cation permeation by cation-pi interaction and prevents anion
permeation by a luminal steric effect. This provides new insight into how ion selectivity
is achieved in the paracellular pore.

Claudin-2
Claudin-10b
Claudin-15
Claudin-16
Claudin-17
Claudin-10a
Claudin-4
Cation pore
Cation pore
Cation pore
Cation pore
Anion pore
Anion pore
Anion pore
C A T H S T - G I T Q C D I Y S T
C V T D S T - G V S N C K D F P S
C A T D S L - G V Y N C W E F P S
C V T N A F D G I R T C D E Y D S
C V V QS T - G QMQ C K V Y D S
C A GN A L - G S F H C R P H F S
C I R QA R - V R L Q C K F Y S S
0+1 +3 +2 +4
Figure 3-7. Homology alignment of major pore-forming claudins.
Cation-selective pore claudins: claudin-2(63), claudin-10b(22, 45, 58), claudin-15(51), claudin-16(25).
Anion-selective pore claudins: claudin-17(33), claudin-10a(22, 58), claudin-4(26). Claudins with
inconclusive or controversial selectivity properties, such as claudin-7 and claudin-19, are excluded.
Displayed here is a homology alignment of the amino acid sequence of the first extracellular domain
from the first conserved extracellular cysteine to the fifth residue downstream of the second conserved
cysteine. Negatively charged residues are in red, positively charged residues are in blue, and aromatic
residues are in orange. The numbers denote relative positions downstream of the second cysteine, where
0 corresponds to the second conserved extracellular cysteine, +1 corresponds to D65 in claudin-2, and
+3 corresponds to Y67 in claudin-2 and F66 in claudin-10b.

56
CHAPTER 4: COMPREHENSIVE CYSTEINE-SCANNING
MUTAGENESIS REVEALS CLAUDIN-2 PORE LINING RESIDUES
WITH DISTINCT CHARACTERISTICS
ABSTRACT
In tight junctions, the first extracellular loop (ECL1) of claudins forms
paracellular pores that determine the paracellular ion permselectivity. Previous selective
cysteine mutagenesis of claudin-2 identified I66 as a pore-lining residue. We aimed to
map out all pore-lining residues of claudin-2 through comprehensive cysteine-scanning
mutagenesis of ECL1. We initially screened 45 cysteine mutants of the ECL1 in
polyclonal MDCK II Tet-off cells, and found that nine mutants displayed a significant
decrease of conductance after treatment with a thiol-reactive reagent, (2-
(trimethylammonium)ethyl methanethiosulfonate (MTSET). This suggests them being
the candidates of the pore-lining residue. Next, we expressed these candidates in
monoclonal MDCK I Tet-off cells for confirmatory studies. All mutants had similar ion
permselectivity and pore size as wild-type claudin-2. Nevertheless the conductance
inhibition assay of a panel of MTS reagents revealed distinct pore-blocking effects. Both
positively charged and neutral MTS reagents inhibited the conductance of S68C, S47C,
I66C, and T32C, while only the positively charged reagents inhibited the conductance of
T56C, T62C, M52C, and G45C. For residues within the narrow part of the pore, such as
in S68C, S47C, and T62C, where free diffusion of MTS molecules is restricted, the

57
kinetics of reaction with MTS molecules decreased disproportionally as the size of the
molecule increased. In conclusion, we describe, for the first time, the full claudin-2 pore
region and the distinct geometrical location of each pore-lining residue. This can be
applied to future x-ray crystal structures and molecular modeling of claudins to further
understand the molecular mechanism for paracellular ion transport.
INTRODUCTION
In epithelia, tight junction is the most apical adhesion complex and constitutes the
paracellular barrier. Ions can be transported across the epithelia via both transcellular and
paracellular pathways. Most transcellular ion channels are formed by amphipathic
transmembrane α-helixes, and the hydrophilic side of the helixes lines up the ion-
selective filter. In contrast, paracellular ion transport occurs in the extracellular space
parallel to the lateral membrane of the cells. A family of tight junction tetra-
transmembrane proteins known as claudins determines the paracellular ion
permselectivity (46). The first extracellular loop of claudins forms paracellular ion pores
(6), by the interaction of two opposing claudin molecules on two adjacent cells, as well as
by the interaction of two neighboring extracellular loops within one cell. Because of the
aqueous nature of the paracellular pore, the pore structure and the molecular mechanism
for ion selectivity are believed to be unique compared to transcellular channels and
transporters.
Claudin-2 forms a paracellular cation pore. The pore is 6.5~7.5 Å in diameter.
Cations permeate through the pore in a partially dehydrated form. The energy for

58
dehydration is primarily gained from the electrostatic interaction between an intrapore
residue D65 and the permeating cation (63). These observations prompt us to hypothesize
that the claudin-2 pore is a cylindrical channel lining by a group of residues, including
D65. The substituted cysteines accessibilities method is commonly used to map the pore-
region of ion channels by showing inhibition of pore function when thiol-reactive
reagents modify the substituted cysteines in the pore-region (14, 32, 47). A selective
cysteine mutagenesis in four positions of claudin-2 identifies I66, a neighboring residue
of D65, as a pore-lining residue (5). Although this residue is adjacent to the intrapore ion-
binding site D65, there is insufficient information to identify any structural pattern. We
aim to map out all the pore-lining residues of claudin-2 by comprehensive cysteine-
scanning mutagenesis of the first extracellular loop. This study would provide new
insight as to the structure-function of claudin-2 pore, and by homologous alignment,
could be generalizable to the ion selectivity mechanism of other claudins.
MATERIALS AND METHODS
Generation of 45 polyclonal MDCK II Tet-off cell lines transduced with cysteine
mutations of the first extracellular loop (ECL1) of claudin-2. MDCK II cells were chosen
to establish polyclonal cell lines because they have a more stable and homogenous
epithelial morphology than MDCK I cells. R31 to A82 were predicted to constitute the
ECL1 of mouse claudin-2 by TMHMM Server v2.0. Each residue (excluding the two
conserved cysteines: C54 and C64, and five residues already mutated to cysteine: Y35,
H57, D65, I66, and Y67) was changed to cysteine using de novo synthesis (GeneScript)

59
and cloned into the pRevTREP vector. 45 plasmids were lipofected into the viral
packaging cell line PT67 on a 96-well plate, and stable clones were selected under
0.3mg/ml hygromycin for 7-10 days. Viral particles were collected from the growth
medium of the PT67 cells and used to transduce MDCK II Tet-off cells. After 7-10 days
in a 0.3 mg/ml hygromycin selective medium, stably transduced polyclonal MDCK II
Tet-off cell lines were generated.
Immunoblotting. The protein expression of claudin-2 mutants was tested by SDS-
PAGE and immunoblotting. Confluent cells grown on tissue culture dishes were
mechanically lysed by passing through a 25-gauge needle 10 times in a sucrose-histidine
lysis buffer containing 0.25M sucrose, 30mM histidine, 1mM EDTA and pH 7 with
protease inhibitor (Complete Mini, Roche Diagnostics). Cell lysates were loaded in a
reducing SDS-PAGE buffer (1% (v/v) 2-mercaptoethanol added) and heated at 75˚C for
10 minutes. 20µg of protein samples were loaded into 12% polyacrylamide gel,
transferred to a PVDF membrane, blotted with 1:500 mouse anti-claudin-2 antibody (Life
Technologies) and 1:1000 rabbit anti-β-actin antibody (Life Technologies), and
appropriate horseradish peroxidase-conjugated secondary antibodies (GE), detected with
the chemiluminescent method (Pierce), and imaged by an ImageQuant LAS-4000 (GE
Healthcare). Because MDCK II Tet-off cells had endogenous claudin-2 expression, the
total claudin-2 expression in both uninduced conditions (Dox+) and induced conditions
(Dox-) was normalized to its β-actin expression. The fold increase of claudin-2

60
expression, calculated as X=(Cldn2
D-
/Cldn2
D+
)/(Actin
D-
/Actin
D+
), represented the
expression level of exogenous claudin-2 mutants.
Immunofluorescent staining. Cells were plated at a confluent density on a 96-well
glass bottom plate (for MDCK II Tet-off cells) or a density of 10
5
cells/ 1.16 cm
2
on 12-
well Transwell plates (for MDCK I Tet-off cells), and grown for seven days. The cells
were washed in ice-cool phosphate buffered saline (PBS), fixed with 4%
paraformaldehyde (PFA) at 4˚C for 15 minutes, permeabilized and blocked in a
permeation buffer (0.3% Triton X-100, 1% BSA, and 5% goat serum in PBS) for one
hour. The filters were incubated in primary antibodies (mouse anti-claudin-2, 1:500;
rabbit anti-ZO-1, 1:500) for two hours at room temperature, washed with PBS, incubated
in secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor
555-conjugated anti-mouse IgG, both 1:1000) for one hour, and washed with PBS. The
transwell membrane were cut off from the holder and mounted in ProLong anti-fade
mounting medium. All the reagents were acquired from Life Technologies. The 96-well
glass bottom plate was imaged directly by an ordinary immunofluorescent microscope.
Slides were imaged by a Leica TCS SP2 multi-photon confocal microscope.
Measuring the transepithelial resistance of the polyclonal MDCK II Tet-off cell
lines. Cells were plated at a density of 10
5
cells/1.16 cm
2
on 12-well Transwell plates in
uninduced conditions (Dox+) and induced conditions (Dox-). On day six, the filters were
transferred to CellZscope (Nanoanalytics) and equilibrated overnight. On day seven, the
TER was measured at one-hour intervals for 48 hours. The maximum TER during this

61
period was recorded and converted to conductance G (in mS). The folds increase of
conductance Y in each filter was calculated by the following: Y =G
D!
/G
D+
, and
presented as a 95% confidence interval (n=3-6).
Screening assay of pore conductance inhibition by MTSET. Cells were plated at a
density of 10
5
cells/1.16 cm
2
on 12-well Transwell plates in uninduced conditions (Dox+)
and induced conditions (Dox-), transferred to CellZscope on day six, and equilibrated
overnight. On day seven, 100x freshly dissolved (2-(trimethylammonium)ethyl
methanethiosulfonate bromide (MTSET-Br, from Biotium) in PBS was added to the
apical and basolateral compartments of each well to reach a final concentration of 1mM.
The TER was recorded at the zero time point and the one-hour time point, and converted
to G (in mS). The change of conductance was calculated by the following:
!G=(G
1D"
"G
1D+
)"(G
0D"
"G
0D+
) . The ∆G of each mutant was compared to the ∆G of
wild-type (in blue) using one-way ANOVA test with the Bonferroni correction. Residues
with ∆G significantly greater than that of wild-type were defined as the pore-lining
residue (in red). Mutants that did not increase transepithelial conductance (V44C, K48C,
G49C, L50C, W51C, G60C, P74C, A79C) were excluded.
Generation and screening of monoclonal MDCK I Tet-off cell lines expressing
claudin-2 pore lining mutants. MDCK I Tet-off cells expressing claudin-2 pore lining
mutants (T32C, G37C, G45C, S47C, M52C, T56C, S58C, T62C, and S68C) were
generated by methods described previously (62). Independent clones transduced with
claudin-2 constructs were selected using cloning cylinders and were screened by

62
immunoblotting as described above. Clones with high protein expression and epithelial
morphology were chosen.
Electrophysiological studies in Ussing chamber. Cells were plated at a density of
10
5
cells/1.16cm
2
on Snapwell filters (Corning) and cultured for seven days in the
presence (Dox+) or absence (Dox-) of 50 ng/ml doxycycline. The Ussing chamber setup
and liquid junction potential correction method was employed as previously described
(62). The conductance and permeability attributable to the claudin-2 pore was calculated
by subtracting the average value of the uninduced (Dox+) state from the values of the
induced (Dox-) state. The standard Ringer solution used at baseline contained (in mM):
NaCl 150, CaCl
2
2, MgCl
2
1, glucose 10, Tris-HEPES 10, pH 7.4. To measure Na
+

permeability, the solution of the basolateral chamber was changed to a 75mM NaCl
Ringer solution (osmolarity adjusted with mannitol). To measure alkali metal biionic
potential, the basolateral medium was changed to a 150mM alkali metal chloride salt. To
measure organic cation permeability, the basolateral medium was changed to a
combination of 75mM organic cation chloride salt and 75mM NaCl. The organic cations
included methylamine (MA), ethylamine (EA), tetramethylammonium (TMA),
tetraethylammonium (TEA). The ion permeability β=P
Cl
-
/P
Na
+
was calculated from the
Goldman-Hodgkin-Katz voltage equation. The absolute Na
+
permeability was estimated
by the method devised by Kimizuka and Koketsu(63). The alkali metal permeability was
calculated from γ=P
M
/P
Na
, where γ was estimated as (1):
! = (1+")•e
v/
RT
F
!" (1)

63
The organic cation permeability was calculated from the following equation:
! ="(1+#)•e
V/
RT
F
!"#!1 (2)
Here, α represents the activity ratio of NaCl in the apical compartment over the
basolateral compartment. The activity of NaCl at 150mM is 0.752, and 0.797 at 75mM,
resulting in α =(150*0.752)/(75*0.797) = 1.89.
The pore size of claudin-2 mutants was estimated by the method previously
described(63). In short, the claudin-2 pore was assumed to be a cyclinder of diameter, D,
across which cations of diameter, d, diffused. According to the Renkin equation:
P=
A
d
1!
d
D
"
#
$
%
&
'
2
(3)
The square root of the relative permeability of MA, EA, and TEA to Na
+
is linearly
related to the cation diameter. D was estimated from the x-intercept of the best-fit line, as
determined by linear regression.
Confirmatory assay of pore conductance inhibition by a panel of MTS reagents. A
panel of MTS reagents of different charges and sizes, including: 2-aminoethyl
methanethiosulfonate (MTSEA), 2-(trimethylammonium)ethyl methanethiosulfonate
(MTSET), 3-(Triethylammonium)propyl methanethiosulfonate (MTS-PTrEA), and 2-
((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin), were used to test the pore
conductance inhibition effect on the pore-lining residues and the kinetics of reaction. The
working concentrations of the reagents are summarized in Table 4-3. Cells were grown
on Snapwell (Corning) for seven days before being mounted on Ussing chamber. The

64
stock solution was added simultaneously to the medium of the apical and basolateral
chambers and rapidly mixed by gas lifts. The conductance was monitored at one-second
intervals for five minutes except where stated otherwise. The %ΔG was calculated as:
%!G=100%"(G
120s
#G
0
)/G
0
. The decrease of conductance over time was modeled as a
one-phase decay model (by Prism 6.0). The rate constant (K) derived from the curve
represents the kinetics of reaction of the MTS reagent with the substituted pore-lining
cysteines. In a free diffusion state, the rate of reaction is proportional to the rate of
diffusion. A molecule’s rate of diffusion is inversely proportional to its friction
coefficient, which depends on both size and shape of the molecule. Assuming, for the
sake of simplicity, that the shape of MTS molecules is a sphere, the rate of diffusion is
inversely proportional to the radius of the diffusing molecule, and hence inversely
proportional to the cube root of molecular weight. The K’
MTSET
in free diffusion state is
calculated as: K'
MTSET
=K
MTSEA
!
MW
MTSEA
MW
MTSET
3 , to which the K
MTSET
in actual
experimentation is normalized.
Statistics. The data are presented as mean ± standard error. Statistical significance
was determined using unpaired two-tailed Student’s t-test or one-way ANOVA test. The
P value of the multiple comparisons was adjusted using the Bonferroni correction. P<
0.05 was considered to be statistically significant, except where stated otherwise.

65
RESULTS
Characterization of polyclonal MDCK II Tet-off cell lines expressing each cysteine
mutant.
We generated 45 cysteine mutants of the first extracellular loop of claudin-2 (ECL1)
in stably transduced polyclonal MDCK II Tet-off cells by retroviral transduction. First,
the protein expression of transduced claudin-2 constructs was determined by
immunoblotting (Figure 4-1). Since MDCK II Tet-off cells have endogenous claudin-2
expression, we quantified the level of expression of the exogenous claudin-2 as the fold
increase of protein expression (Table 4-1). 41 out of 45 mutants (except R31C, T32C,
E53C, and A79C) had more than a two-fold increase in protein expression.
Next, claudin-2 localization was examined by immunofluorescent staining with a
claudin-2 antibody (Figure 4-2) and summarized in Table 1. The following cysteine
mutants were localized intracellularly: G49C, L50C, W51C, G60C, and P74C. The
mutation to the conserved motif “G
49
L
50
W
51
” resulted in claudin-2 mislocalization, which
is consistent with others’ observation (10, 57). The mislocalization of G60C and P74C
was described for the first time. Since glycine and proline are often found at the turn of a
peptide fold, G60 and P74 may be important in forming the critical turning points of the
claudin-2 ECL1 protein fold. The localization for K48C and E53C were inconclusive due
to the low level of expression.

66

R31C T32C S33C S34C
Dox + - + - + - + -
Cldn2
Actin
+- + - + - + -
V36C G37C A38C S39C
I40C V41C T42C A43C
+- +- +- +- + - +- +- +-
V44C G45C F46C S47C
Dox
Cldn2
Actin
K48C G49C L50C W51C
+- +- +- +- + - +- +- +-
A82C E53C A55C T56C
Dox
Cldn2
Actin
S58C T59C G60C I61C
+- +- +- +- + - +- +- +-
T62C Q63C S68C T69C
Dox
Cldn2
Actin
L70C L71C G72C L73C
+- +- +- +- + - +- +- +-
P74C A75C D76C I77C
Dox
Cldn2
Actin
Q78C A79C A80C Q81C
+- +- +- +- + -
M52C
Dox
Cldn2
Actin
Figure 4-1. Screening of protein expression in polyclonal MDCK II Tet-off cells expressing 45 single
cysteine mutants.
Cell lysates were subjected to reducing SDS-PAGE gel and immunoblotted with anti-claudin-2 antibody
and anti-β-actin antibody. The total claudin-2 expression in both uninduced conditions (Dox+) and
induced conditions (Dox-) was normalized to its β-actin expression. The increase of claudin-2
expression, calculated as X=(Cldn2
D-
/Cldn2
D+
)/(Actin
D-
/Actin
D+
), represented the expression level of
exogenous claudin-2 mutants.

67

Lastly, the transepithelial conductance of the exogenous claudin-2 mutants was
screened in CellZscope under uninduced (Dox+) and induced (Dox-) conditions, and
Figure 4-2. Screening of protein localization in polyclonal MDCK II Tet-off cells expressing 45 single
cysteine mutants.
Cells were plated at confluent density on a 96-well glass bottom plate and grown for seven days. The
cells were stained with primary antibodies against claudin-2 (green). The 96-well glass bottom plate was
imaged directly by a regular immunofluorescent microscope at the exposure time from 2-5 seconds.

68
normalized as fold increase of conductance in a 95% confidence interval (Table 4-1).
Note that S34C and G37C increased the baseline conductance approximately 1.5-fold and
3-fold respectively, but immunofluorescent staining (Figure 4-2) indicated them to be
localized intracellularly. This discrepancy could be due to a sampling error of an
immunofluorescent image and warrants an additional future immunofluorescent staining.
The conductance of V44C, K48C, G49C, L50C, W51C, G60C, L71C, P74C, and A79C
in the induced state had no significant increase in conductance, indicating that these
mutants may not have functional claudin-2 pores at the tight junction, presumably due to
protein mislocalization (G49C, L50C, W51C, G60C, and P74C), or low level of
expression (A79C), or protein misfolding (V44C, K48C, L71C). These mutants were
excluded from the screening assay of pore conductance inhibition by MTSET.
Table 4-1. Biological and Physiological Characterization of 45 Cysteine Mutants of
Claudin-2 ECL1 in Polyclonal MDCK II Tet-off Cells
Mutant Protein Localization¶ Protein Expression† Transepithelial Conductance*
R31C TJ 2.0 [1.19, 1.34]
T32C TJ 1.6 [2.04, 2.10]
S33C TJ 2.0 [1.49, 1.68]
S34C Inconclusive 2.1 [1.62, 1.94]
V36C TJ 4.5 [1.08. 1.34]
G37C Inconclusive 3.7 [3.04, 3.26]
A38C TJ 3.5 [1.41, 1.66]

69
S39C TJ 3.6 [2.10, 2.23]
I40C TJ 5.9 [2.35, 2.59]
V41C TJ 5.1 [1.35, 1.54]
T42C TJ 4.2 [1.42, 1.65]
A43C TJ 2.8 [1.42, 1.56]
V44C TJ 4.8 [0.81, 0.95]
G45C TJ 3.2 [1.69, 1.92]
F46C TJ 7.0 [1.46, 177]
S47C TJ 2.1 [1.42, 1.67]
K48C Inconclusive 2.8 [0.97, 1.06]
G49C Intracellular 3.8 [0.96, 1.08]
L50C Intracellular 3.1 [0.79, 1.22]
W51C Intracellular 4.6 [0.94, 1.02]
M52C TJ 3.2 [1.48, 1.74]
E53C Inconclusive 1.8 [1.35, 1.37]
A55C TJ 3.9 [1.55, 1.91]
T56C TJ 3.5 [1.51, 1.68]
S58C TJ 4.6 [1.18, 2.06]
T59C TJ 2.8 [1.23, 1.59]
G60C Intracellular 3.9 [0.89, 0.98]
I61C TJ 4.3 [2.08, 2.21]
T62C TJ 2.9 [2.10, 2.54]
Q63C TJ 3.6 [1.09, 1.10]
S68C TJ 2.9 [1.08, 1.21]
T69C TJ 5.5 [1.32, 1.49]

70
L70C TJ 10.6 [1.14, 1.23]
L71C TJ 21.2 [0.89, 1.04]
G72C TJ 4.0 [1.57, 1.62]
L73C TJ 7.9 [1.14, 1.56]
P74C Intracellular 4.5 [0.71, 1.28]
A75C TJ 7.6 [1.06, 1.15]
D76C TJ 4.1 [1.31, 1.68]
I77C TJ 4.2 [1.29, 1.51]
Q78C TJ 5.2 [1.67, 1.71]
A79C TJ 1.3 [0.93, 1.26]
A80C TJ 2.6 [1.09, 1.19]
Q81C TJ 6.3 [1.16, 1.34]
A82C TJ 2.8 [1.55, 1.74]
¶ TJ: tight junction. Inconclusive: the result is inconclusive because of low expression levels or protein
localization being inconsistent with the functional data.
† Calculated as fold increase of protein expression: X=(Cldn2
D-
/Cldn2
D+
)/(Actin
D-
/Actin
D+
)
* Calculated as fold increase of transepithelial conductance: Y =G
D!
/G
D+

Data presented as 95%
confidence interval of mean.
Screening assay of pore conductance inhibition by MTSET identifies candidates of
pore-lining residue.
One way to identify residues that are located at or close to the pore-lining region
is by showing that when these residues are mutated to cysteine, the thiol-reactive reagent,
MTSET, can inhibit their transepithelial conductance. In the uninduced state for most cell

71
lines, MTSET may change 5-10% of the baseline conductance (data not shown), possibly
due to affecting the transcellular transport. In the induced state of wild-type claudin-2,
MTSET also had a small conductance inhibition effect of 0.5 mS (constituted less than
5% of change, data not shown), possibly because of a non-covalent interaction between
the MTS molecule and the claudin-2 pore. The ΔG of each mutant was compared to the
∆G of wild-type (Figure 4-3, in blue) using one-way ANOVA test with Bonferroni
correction. T32C, G37C, G45C, S47C, M52C, T56C, S58C, T62C, and S68C (Figure 4-
3, in red) had a significantly higher degree of conductance inhibition than wild-type
(Figure 4-3, in blue). These were defined as the pore-lining residues for further
investigations. Some residues had a ΔG greater than that of wild-type (S33C, S34C,
A43C, F46C, Q63C, T69C) but were not statistical significant, possibly because of the
stringent statistical method. They could be used as the second cohort for future study.
Some residues (R31C, S39C, T42C, A55C, I77C, Q78C) had a slightly increase of
conductance after MTSET treatment, perhaps due to an off-target effect on transcellular
transport. Residues that did not exhibit a change of conductance could be explained by its
absence in the pore region or being inaccessible to MTSET.

72

Biochemical and electrophysiological characterization of monoclonal MDCK I Tet-
off cell lines expressing the pore-lining residues.
To fully characterize the pore properties of each pore-lining residue, we generated
monoclonal MDCK I Tet-off cell lines expressing the respective cysteine mutants (except
G37C and S58C, which have not yet been generated). Inducible protein expression was
verified by immunoblotting (Figure 4-4A), which showed a characteristic band of
claudin-2 monomer at approximately 20kD in the absence of doxycycline. There were
also multiple bands smaller than 20kD, which are not seen in the claudin-2 blot of mouse
kidney lysates (data not shown). They are therefore probably proteolysis products, which
Screening: Conductance of claudin-2 pore after MTSET
WT
R31C
T32C
S33C
S34C
V36C
G37C
A38C
S39C
I40C
V41C
T42C
A43C
G45C
F46C
S47C
M52C
E53C
A55C
T56C
S58C
T59C
I61C
T62C
Q63C
S68C
T69C
L70C
G72C
L73C
A75C
D76C
I77C
Q78C
A80C
Q81C
A82C
-4
-3
-2
-1
0
1
** *
*** *** *** *** *** *** ***
Wild type claudin-2 Pore-lining residue candidates
Conductance (mS)
Figure 4-3. Screening assay of conductance inhibition by MTSET.
Cells were cultured in Dox+ and Dox- for seven days. The transepithelial resistant (TER) was
monitored in CellZcope at one-hour intervals. After the TER reached a steady state, 1mM MTSET was
added to the wells. The TER was recorded at zero and one-hour time points. ΔG=(G
1Dox-
– G
1Dox+
)-
(G
0Dox-
– G
0Dox+
). The statistical significance was determined by one-way ANOVA with the Bonferroni
correction. *p<0.05, **p<0.01, ***p<0.001.

73
we often see in overexpressing proteins in cells. Immunofluorescent staining with anti-
claudin-2 (green) and anti-ZO-1 (red) antibodies shows that each pore-lining candidate
had similar tight junction localization as wild-type claudin-2 (Figure 4-4B).

Figure 4-4. Characterization of stably transfected MDCK I Tet-Off cell lines expressing claudin-2
mutants.
(A) Immunoblot of claudin-2 expression in clones stably transduced with wild-type, T32C, G45C,
S47C, M52C, T56C, T62C, I66C, and S68C, grown in the presence (+) or absence (-) of doxycycline
(Dox). Cell lysates were subjected to reducing SDS-PAGE and immunoblotted with claudin-2 antibody.
The image was exposed for 15 minutes. Inducible claudin-2 monomer expression at approximately
20kD was seen in all mutants. (B) Localization of claudin-2 mutants by immunofluorescent staining and
confocal microscopy examination. The cells were cultured on Transwells for six days, then
immunostained for ZO-1 (red) and claudin-2 (green). All mutants co-localized well with ZO-1 at the
tight junction.

74

Next, the eletrophysiological properties of each mutant were analyzed in Ussing
chamber. The increase in conductance ranged from 4-folds (I66C) to 13-folds (M52C)
(Figure 4-5A). The P
Cl
-
was not different among these constructs, and the P
Na
+
was in
accordance with the level of the induced conductance (Figure 4-5B). All mutant pores
Relative permeability
1234567
0.0
0.5
1.0
1.5
2.0
Li
+
Na
+
K
+
Rb
+
Cs
+
MA
+
EA
+
TMA
+
TEA
+
Ionic diameter (Å)
P
M
+
/P
Na
+
Diameter estimation
345678
0.0
0.5
1.0
1.5 WT
T32C
G45C
S47C
M52C
T56C
T62C
I66C
S68C
MA+
EA+
TMA+
Ionic diameter (Å)
Sqaure root of P
M
+
/P
Na
+
AB
CD
Conductance after protein induction
WT
T32C
G45C
S47C
M52C
T56C
T62C
I66C
S68C
0
5
10
15
Dox+
Dox-
Conductance (mS)
Na
+
and Cl
-
permeability
WT
T32C
G45C
S47C
M52C
T56C
T62C
I66C
S68C
0
5
10
15
20
25 PNa
PCl
Permeability (*10
-6
cm/s)
Cation selectivity
WT
T32C
G45C
S47C
M52C
T56C
T62C
I66C
S68C
0
2
4
6
8
10
P
Na
+
/P
Cl
-
E
Figure 4-5. Characterization of the electrophysiological properties of claudin-2 cysteine mutants of the
pore-lining residues.
MDCK I To wild-type claudin-2, T32C, G45C, S47C, M52C, T56C, T62C, I66C, and S68C were
plated at a density of 10
5
cells/1.16cm
2
and grown for seven days before mounting on Ussing Chamber.
(A) The increase of conductance after protein induction (Dox-) (B) Na
+
permeability and Cl
-

permeability determined by 2:1 NaCl dilution potentials. (C) Ion selectivity was calculated as P
Na
+
/P
Cl
-
,
where P
Na+
and P
Cl-

were calculated from NaCl dilution potentials and subtracting the average baseline
permeability of uninduced (Dox+) cells from that of induced (Dox-) cells. (D) The permeability of
wild-type and the pore-lining mutants to alkali metal cations and organic cations relative to their Na
+

permeability were plotted against the ionic diameters. (E) The relationship between the square roots of
the relative permeability of methylamine (MA), ethylamine (EA), and tetramethylammonium (TMA)
and ionic diameters were fitted by linear regression, and the pore diameter was estimated as the x-
intercept of the best-fit line. Data points represent the means of 3-9 filters ± SE

75
were cation selective with the P
Na
+
/P
Cl
-
ranging from 4 (I66C) to 8 (wild type) (Figure 4-
5C). For all mutants, the relative permeabilities of alkali metal cations and organic
cations to the Na
+
permeability were similar to wild-type (Figure 4-5D). The estimated
pore diameter ranged from 6.4 Å to 7.3 Å (Figure 4-5E and Table 4-2), which was not
significantly different from wild-type (6.3Å). In summary, all mutants had similar pore
properties to wild-type, and by inference, they have similar pore structure to wild-type.
Table 4-2. Summary of Estimated Pore Size of Cysteine Mutants and Wild-Type
Claudin-2
Construct Pore diameter (in Å) 95% Confidence Interval R
2

Wild-type 6.3 [5.9, 6.9] 0.9154
T32C 6.6 [6.1, 7.5] 0.9012
G45C 6.6 [6.2, 7.3] 0.8468
S47C 6.4 [6.1, 6.7] 0.9019
M52C 6.5 [6.1, 6.9] 0.9563
T56C 7.3 [6.3, 10.2] 0.7509
T62C 6.4 [6.2, 6.7] 0.9747
I66C 6.4 [6.1, 6.9] 0.9423
S68C 7.0 [6.2, 9,2] 0.7843

76
Pore-lining residues reveal distinct patterns of conductance inhibition by thiol-
reactive reagents.
We hypothesized that thiol-reactive reagents of varying charges and sizes would
block the conductance differently, and that the inhibition pattern may provide information
about the geometrical location of that residue. Residues within the narrow part of the pore
would have more inhibition by MTS molecules than residues located in the wider part of
the pore. Furthermore, residues in the wide part of the pore would be inhibited more by
larger MTS molecules than by smaller MTS molecules (Figure 4-6). The panel of thiol-
reactive reagents used is summarized in Table 4-3.

Figure 4-6. Conductance inhibition reflects pore location.
Upper panel: Residues within the narrow part of the pore would have more inhibition by MTS
molecules than residues located in the wider part of the pore. Lower panel: residues in the wide part of
the pore would be inhibited more by larger MTS molecules than by smaller MTS molecules

77
Table 4-3. Size, Charge, and Working Concentration of The Thiol-Reactive Reagents
Used In This Study*
Chemical Name Abbreviation Net Charge Molecular
weight (g/mol)
Working
Con. (mM)
Stock Con.
2-aminoethyl
methanethiosulfonate
hydrobromide
MTSEA + 236 2.5 100x
[2-
(Trimethylammon
ium)ethyl]
methanethiosulfon
ate Bromide
MTSET + 278 1 100x
3-
(Triethylammoniu
m)propyl
Methanethiosulfo
nate Bromide
MTS PTrEA + 334 1 100x
2-
((biotinoyl)amino)
ethyl
MethaneThioSulfo
nate
MTSEA-
biotin
Neutral 382 1 5x
*Reagents are listed in order of net charge, increasing size

78
As suggested by the previous screening assay, less than 10% change in
conductance was considered as a non-specific effect. I66C showed consistent results with
the previous study (5) and was used as the positive control. The conductance inhibition
revealed distinct patterns (Figure 4-7). Consistent with the screening assay, S68C had the
most conductance inhibition by both the positively charged and neutral MTS molecules.
MTSEA blocked more than 60% of the pore conductance. MTSET, MTS PTrEA and
MTSEA-biotin inhibited the conductance similarly by a degree of 40%. This remarkable
inhibition effect suggests that this site locates very close to the pore selective filter, and
its side chain probably points along the direction where ions pass through. S47C, T56C,
and T62C had approximately 20-30% of conductance inhibition by positively charged
MTS molecules. However, S47C had approximately 15% of conductance inhibition by
MTSEA-biotin, yet in contrast, either T56C or T62C was not inhibited by MTSEA-
biotin. The lack of effect of MTSEA-biotin on T56C and T62C could be because they are
located in a narrow part of the pore, a location that MTSEA-biotin cannot access.
Alternatively, these sites could be located in a wider part of the pore, where MTSEA-
biotin can covalent react with the substituted cysteines but cannot inhibit the pore
conductance. To distinguish between these two hypotheses, we could test whether pre-
incubation of MTSEA-biotin protects these cysteines from being modified by positively
charged MTS reagents and hence blunts the pore-blocking effect. T32C was inhibited by
large MTS molecules (MTS PTrEA and MTSEA-biotin), regardless of the net charge, but
not by the small ones, suggesting it is located in a wide part of the pore. Unlike T32C,

79
G45C was inhibited by small MTS molecules but not by the large ones. This suggests
that the side chain of G45 locates in a hindered position, either in a narrow part of the
pore or hindered by its neighboring residues, or both. M52 displayed the least inhibition
by MTS molecules, indicating that it is located in the widest part of the pore. In
summary, S68C, G45C, S47C, T56C and T62C are likely located in a narrow part of the
pore, as in the case of I66C(5); T32C and M52C are likely located in a wider part of the
pore; G45C likely points to a partially hindered position.

Conductance inhibition by MTS reagents
-80%
-60%
-40%
-20%
0%
MTSEA(+)
MTSET(+)
MTSEA-Biotin (neutral)
S68C S47C I66C T62C T56C M52C
MTSPTrEA(+)
G45C T32C
% conductance
Figure 4-7. Confirmatory assay of conductance inhibition by a panel of thiol-reactive reagents.
Cells were grown on Snapwells for seven days before mounting on Ussing chamber. The stock solution
of MTS reagents was added simultaneously to the medium of the apical and basolateral chambers. The
change in conductance was calculated by as percentage decrease in conductance from pre-treatment
state to the 120-second post-treatment state.

80
Kinetics of reaction of MTS reagents with the substituted pore-lining cysteines.
If the substituted cysteine is hindered, either because it is located within the
narrow part of the pore or because a bulky neighboring residue hinders it, the kinetics of
reaction will be low. Furthermore, if MTS molecules react with the thiol group in a free
diffusion state, the rate constant will be inversely proportional to the size of the molecule.
If the thiol group is in a hindered position, the rate constant will decrease
disproportionally as the size of the MTS molecule increases. The degree of the
disproportion indicates the degree of hindrance (Figure 4-8). Therefore, besides the
degree of inhibition, the kinetics of reaction could provide additional information about
the geometrical location of the pore-lining residues. S47C and G45C had smaller K
MTSEA
than the other residues (Figure 4-9A), suggesting their accessibility is restricted.

81

Figure 4-8. Kinetics of reaction reflects pore location.
Upper panel: in free diffusion state, the rate of reaction of MTS molecules with a thiol group is
proportional to the rate of diffusion. The rate of diffusion is inversely proportional to the radius of the
molecule, and hence to the cube root of the molecular weight. Lower panel: if the MTS molecules need
to diffuse through a narrow channel, the rate of reaction for larger MTS molecule (MTSET) will
decrease disproportionally than the rate of reaction for smaller MTS molecule (MTSEA).

82
When normalized, the individual K
MTSET
to the K’
MTSET
in free diffusion calculated from
the respective K
MTSEA
obtained in the experiment, The K
MTSET
of S68C, S47C, and T62C
decreased disproportionally (the ratio should equal one if MTSET diffuses freely through
the pore), despite the fact that the p value of S68C and T62C did not reach statistical
significance (Figure 4-9B). The K
MTSET
of other residues decreased disproportionally to
some extent, but none were statistically significant.
MTSEA and MTSET
I66C
S68C
S47C
T62C
T56C
T32C
G45C
0
50
100
150
MTSEA MTSET
Kinetics constant (M
-1
*S
-1
)
Normalized MTSET
I66C
S68C
S47C
T62C
T56C
T32C
G45C
0.0
0.5
1.0
1.5
MTSEA MTSET
p=0.054 p<0.0001 p=0.077
Ratio
AB
Figure 4-9. Kinetics of reaction of MTS reagents with the substituted cysteines.
The decrease in conductance over time was modeled as a one-phase decay model (by Prism 6.0). The
rate constant (K) derived from the curve represents the kinetics of reaction of the MTS reagents with the
substituted pore-lining cysteines. The K’
MTSET
in free diffusion state is calculated in the following way:
K'
MTSET
=K
MTSEA
!
MW
MTSEA
MW
MTSET
3 , to which the K
MTSET
in actual experiment is normalized.

83
DISCUSSION
Claudin-2 is a paracellular cation pore that determines the paracellular ion
permselectivity. Previous works show that both D65 (63) and Y67 (manuscript in review)
operate as the sites conferring cation selectivity, and I66 lines up the pore lumen (4). The
composition of the pore region is incompletely understood. This work aims to map out
the full pore-lining region of claudin-2 by comprehensive cyseteine-scanning
mutagenesis and thiol modification. Conventionally, pore-forming claudins are studied in
monoclonal epithelial cell lines of low baseline conductance, such as MDCK I cells, to
obtain a high signal-to-noise ratio and experimental consistency (62). But this method is
not practical for generating 45 mutants. Instead, we profiled the role of each residue of
ECL1 using polyclonal MDCK II Tet-off cells, followed by confirmatory studies of the
pore-lining residues in monoclonal MDCK I Tet-off cells. The screening assay of
conductance inhibition by MTSET, coupled with a stringent statistical method, found that
at least nine new residues (T32, G37, G45, S47, M52, T56, S58, T62, and S68) are lining
the pore. This may exclude some potential pore-lining residues, such as S33C and S34C
(close to T32), A43C (close to G45), F46C (close to G45 and S47), Q63C (close to T62),
T69C (close to S68), but we believe that the neighboring pore-lining residues of those
that are excluded may provide sufficient structural-functional information about the sub-
regions of the pore.
The screening assay has limitations. First, MDCK II cells endogenously express
claudin-2. The claudin-2 pore is a multimeric pore composed of endogenous claudin-2

84
and claudin-2 cysteine mutant in various ratios. The number of substituted cysteines in
each pore varies. Second, the pore properties of the mutants cannot be accurately
determined because of the high baseline conductance of MDCK II cells and hence a low
signal-to-noise ratio. Third, CellZscope only measures transepithelial resistance at
intervals of an hour. This does not provide the kinetics of reaction. Therefore, the pore-
lining residues were generated in monoclonal MDCK I Tet-off cells, which lacks
endogenous claudin-2, for a comprehensive characterization of pore function and
cysteine modification.
The seven pore-lining residues expressed in MDCK I cells displayed similar ion
permselectivity and pore size. The P
Na
+
of some clones differed from wild-type claudin-2,
in accordance with the variation in conductance in each clone, probably related to the
level of claudin expression at the tight junction. These residues, although lining the pore,
are not directly involved in the mechanism of ion permeation and that their substitutions
do not lead to change in pore conformation. The residues, however, displayed very
different accessibility and kinetics of reaction to thiol-reactive reagents, which provide
indirect structural information of the pore.
Assuming the claudin-2 pore is a cylindrical channel, the entry of the pore is
relatively wide, and the selectively filter is the narrowest part. T32 and M52 are believed
to be located in the wider part of the pore because they have a small inhibition effect by
small MTS molecules and significantly larger blockage effect by larger MTS molecules.
The degree of conductance blockage reflects the degree of proximity of that residue to the

85
selective filter, suggesting that S68 is located narrowest part of the pore. However, this
model may be over-simplified. In fact, the degree of blockage is related to the width of
molecule that is projected on the cross-sectional plane of the selective filter. This is a
function of four factors: the size of the molecules, the flexibility of the spacer arm of the
molecules, the distance of the side chain to the cross-sectional plane of the selective filter,
and the angle between the side chain and the cross-sectional plane of the selective filter.
This explains why S68C is further downstream of I66C yet has more inhibition by MTS
reagents. It also explains why the smallest MTSEA has a larger blockage effect in S68C
than other larger molecules. This is probably because MTSEA binding to S68C yields the
largest projectional component onto the cross-sectional plane of the selective filter.
Nevertheless, S68, along with G45, S47, T56, T62, and I66, is believed to be located in a
narrower part of the pore than where T32 and M52 are located. Note that the lack of
effect of MTSEA-biotin on T56C and T62C could be because they are located in a
narrow part of the pore, where MTSEA-biotin cannot access, or because they could be
located in a wider part of the pore where MTSEA-biotin can covalent react with them but
cannot inhibit the conductance. This requires further study.
The kinetics of reaction provides an extra layer of information to distinguish the pore
location. If the MTS reagent reacts with the free cysteine in a free diffusion state, the rate
of reaction is proportional to the rate of diffusion. If the diffusion of the molecules is
restricted because of a narrow pathway, or if the accessibility of the cysteine is partially
blocked by steric hindrance from neighboring side chains, the kinetics of reaction will be

86
low. The K
MTSEA
of G45C and S47C are lower than others, probably because their
neighboring residue is a phenylalanine (F46), which has a bulky side chain. Furthermore,
assuming, for the sake of simplicity, that the shape of MTS molecules is a sphere, the rate
of diffusion is inversely proportional to the radius of the diffusing molecule, and hence
inversely proportional to the cube root of molecular weight. If the thiol group is in a
hindered position, the rate constant will decreased disproportionally as the size of the
MTS molecule increases. The degree of the disproportion, as reflected by the ratio of the
experimental K
MTSET
versus the K’
MTSET
in free solution (calculated from K
MTSEA
),
indicates the degree of hindrance. In addition, because the K
MTSET
is normalized to the
K
MTSEA
, this eliminates the factor of local accessibility of the side chain and solely
reflects the diffusion of MTSET through the pore, as an indicator for the pore
narrowness. S68C, S47C, and T62C have a more significant disproportional decrease of
K
MTSET
than that of I66C, T56C, T32C, and G45C. The restricted accessibility of S47C is
probably the result of the localization in the narrow part of the pore and the local steric
blockage. S68, S47, and T62 are probably located closer to the selective filter than I66
and T56. In summary, to the best of our understanding of these data, the pore-lining
residues, in the order of proximity to the narrowest part of the pore, are as follows: S68,
S47, T62, I66, T56, T32, G45, and M52. In figure 4-10, We speculate that one structural
element of the pore region is a loop formed by the conserved cysteines because this loop
has at least three residues (T56, S58, T62) that face the pore lumen. Another structural
element is a beta-sheet consisted by the four residues downstream of C64 (D65-S68)

87
because this four residues form a continuous side facing to the pore lumen. So far, the
other pore-lining residues T32, G37, S47, and M52 do not have a clear structural pattern,
but at least, S47 is believed to localize in the narrow part, and T32 and M52 are located in
the wide part.

In conclusion, this study provides a complete map of claudin-2 pore region and
shows that each pore-lining residue has distinct geometrical location within the pore. This
knowledge can be applied to future x-ray crystal structures and molecular modeling of
claudins to further understand the molecular mechanism for paracellular ion transport.

Figure 4-10. Speculative model of pore region.
The two conserved cysteines are linked by a disulfide bond and form a loop. D65 is the major ion-
binding site and Y67 is the minor site for cation selectivity. All residues in blue are pore-lining residues.
We speculate that the pore region consists of at least two structural elements: a loop and a beta-sheet.
T56, S58, and T62 are on the loop and four continuous residues downstream of C64 (D65-S68) are face
the pore lumen, suggesting a beta-sheet structure.

88
CHAPTER 5: SUMMARY AND FUTURE DIRECTION
SUMMARY
This dissertation describes a structure-function study of the paracellular ion pore.
We initially started by accessing the roles of conserved residues in the first extracellular
loop (ECL1) of claudin-2, which is the pore-forming domain, and then scaled up by
conducting a comprehensive cysteine-scanning mutagenesis of the first extracellular loop
and testing the accessibility of the substituted cysteines by thiol-reactive reagents. The
long-term goal of this work is to determine the structural-functional determinants of ion
permselectivity of paracellular ion transport.
In this dissertation, we have made three major discoveries:
1. The extracellular conserved cysteines of ECL1 form an intramolecular disulfide
bond that is necessary for pore function in claudin-2. This finding suggests that the
correct folding and hence proper function of ECL1 requires the two conserved
cysteines to form an intramolecular disulfide bond.
2. The conserved aromatic residue located 1-2 residues downstream of the major
charge selective site has a dual role for cation selectivity. It facilitates cation
permeation by cation-pi interaction and prevents anion permeation by a luminal
steric effect. This provides new insight into how ion selectivity is achieved in the
paracellular pore.

89
3. We map out the full pore region of the claudin-2 using cysteine-scanning
mutagenesis and the substituted cysteine accessibility method. We describe, for the
first time, the full claudin-2 pore region and the distinct geometrical location of each
pore-lining residue. This can be applied to future x-ray crystal structures and
molecular modeling of claudins to further understand the molecular mechanism for
paracellular ion transport.
OVERALL SIGNIFICANCE OF THIS DISSERTATION
Understanding of the structure-function relationship of claudin based paracellular
pore may eventually lead to identify diseases that are due to abnormal paracellular
permeability and elucidate the pathophysiological mechanisms of those diseases,
including salt-sensitive hypertension and tubulopathies that cause electrolytes and acid-
base disorders. Also, understanding the functional role of the residues in the claudin pore-
forming domain may help to identify the relationship of claudins polymorphism and
susceptibility of common diseases related to claudins, such as nephrolithiasis(53) and
pancreatitis(60). Lastly, the knowledge of the pore-lining region can be applied to future
x-ray crystal structures and molecular modeling of claudins to further understand the
molecular mechanism for paracellular ion transport.
FUTURE DIRECTIONS:
The comprehensive cysteine-scanning mutagenesis of claudin-2 ECL1 provide
rich resource for future structural-functional studies.

90
Claudin-claudin interface
Claudins are believed to polymerize within the tight junction, and to be stabilized
by side-by-side interactions between claudin protomers within the same cell and same
tight junction strand (cis-interaction) and by head-to-head interactions between claudins
from the lateral plasma membranes of two adjacent cells (trans-interaction). Numerous
studies show that claudins have both homotypic and heterotyptic interactions at the tight
junction (11, 18, 34, 43). Claudin-claudin interaction has been suggested to promote
breast cancer cell metastasis to the liver(49). The knowledge of claudin-claudin
interaction interface would benefit the drug design to block claudins related cancer
metastasis. The composition of the interaction interface is largely unknown. One way in
which we have been able to identify residues that are located at or close to an
intermolecular interface between claudin proteins is by showing that they form an
intermolecular disulfide bond when they are mutated to cysteine, for example D65 in
claudin-2(5). Using the cysteine mutants of chapter 4, we could map out the interaction
interface of claudn-2.
Structural modeling and stimulation of claudins
In the recent years, bioinformatics-based computational methods have advanced
in three-dimensional (3D) protein structural modeling. The knowledge of pore-lining
region and interaction interface of claudins would provide functional evidence to refine
computational structural models of claudins. In addition, the interaction interface would
enable the docking stimulation to study claudin-claudin interaction. The computational

91
model would enable further understanding of the molecular mechanism for paracellular
ion transport.

92
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Abstract (if available)

Abstract Claudins constitute a family of tight junction transmembrane proteins whose first extracellular loop (ECL1) determines the paracellular permeability and ion selectivity in epithelia. Claudin-2 forms a paracellular cation pore. We are interested in the molecular mechanism of ion selectivity of claudin-2 from a structural-functional perspective. In chapter 2, we explored the role of two highly conserved cysteines in ECL1 by hypothesizing that these extracellular cysteines are linked by an intramolecular disulfide bond. We found that the single cysteine mutants can form a claudin-2 homodimer, suggesting that the two conserved cysteines normally form an intramolecular disulfide bond in wild-type claudin-2. We also found that the disulfide bond is necessary for pore formation. In chapter 3, we tested the role of a highly conserved aromatic residue near the pore selectivity filter of claudins by hypothesizing that it contributes to cation selectivity by cation-pi interaction with the permeating cation. The Y67L mutant showed reduced cation selectivity compared to wild-type claudin-2 due to the decreased Na⁺ permeability, without affecting the Cl⁻ permeability. The Y67A mutant enlarged the pore size and further decreased the charge selectivity due to an increase in Cl⁻ permeability. The Y67F mutant restored the Na⁺ permeability, Cl⁻ permeability, and pore-size back to wild-type. We conclude that the conserved aromatic residue near the cation pore domain of claudins contributes to cation selectivity by a dual role of cation-pi interaction and a luminal steric effect. In chapter 4, we aimed to map out all pore-lining residues of claudin-2 through comprehensive cysteine-scanning mutagenesis of ECL1. We screened 45 cysteine mutants of the ECL1 in polyclonal MDCK II Tet-off cells and found nine pore-lining residues. Next, we stably expressed these candidates in monoclonal MDCK I Tet-off cells for confirmatory studies. The mutants had similar ion permselectivity and pore size as wild-type claudin-2. Nevertheless the conductance inhibition assay of a panel of MTS reagents revealed distinct patterns of blockage effect and varying kinetics of reaction. In conclusion, we identified all pore-lining residues of claudin-2 with distinct geometrical location. This can be applied to future x-ray crystal structures and molecular modeling of claudins to further understand the molecular mechanism for paracellular ion transport.

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Creator Li, Jiahua (author)

Core Title Structural-functional study of the paracellular ion pore

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Systems Biology and Disease

Publication Date 07/23/2013

Defense Date 05/24/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag claudin,ion channel,ion transport,OAI-PMH Harvest,structure-function,tight junction

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Advisor Yu, Alan S. L. (committee chair), Farley, Robert A. (committee member), Langen, Ralf (committee member), McDonough, Alicia A. (committee member), Peti-Peterdi, Janos (committee member)

Creator Email jiahuali@usc.edu,lijiahua1984@gmail.com

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