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GATING MECHANISMS OF TRANSIENT RECEPTOR POTENTIAL ION CHANNELS TRPM5 and TRPM4 IN NATIVE AND HETEROLOGOUS CELLS by Zheng Zhang A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (NEUROSCIENCE) December 2007 Copyright 2007 Zheng Zhang
|Title||Gating mechanisms of transient receptor potential ion channels TRPM5 and TRPM4 in native and heterologous cells|
|Degree||Doctor of Philosophy|
|School||College of Letters, Arts and Sciences|
|Advisor (committee chair)||Liman, Emily R.|
|Advisor (committee member)||
Chow, Robert H.
McKemy, David D.
McDonough, Alicia A.
|Abstract||There are 28 mammalian members of the Transient Receptor Potential (TRP) family of ion channels. Two closely related members TRPM4 and TRPM5 have been shown to be activated by elevation of intracellular Ca2+ in heterologous expression system. TRPM5 is an essential part of the taste signaling pathway, and understanding regulation of this channel is critical to understanding taste sensation. TRPM4 is more broadly expressed and plays a role in mast cell degranulation as well as in many other physiological processes.; In the first part of this thesis, I describe a mechanism by which TRPM4 channels desensitize following activation. My experiments show that heterologously expressed TRPM4 channels desensitize rapidly following exposure to intracellular Ca2+, and that this desensitization involves a change in the gating. I show that channel activity can be recovered by conditions that are expected to elevate PIP2 levels, suggesting that depletion of PIP2 by Ca2+-dependent PLCs may be the mechanism of desensitization. In further experiments I show that the 'TRP box' or 'TRP domain' is unlikely to be the binding domain for PIP2 in TRPM4 channels. I also establish for the first time that a new synthesized non-hydrolyzable PIP2 analog can mimic the activity of native PIP2.; In the second part of this thesis, I take advantage of TRPM5 knockout mice and TRPM5 transgenic mice expressing GFP driven by TRPM5 promoter, to study the mechanism of activation of native TRPM5 channels. My results show for the first time that TRPM5 channels in taste cells are activated by an elevation of IP3 and the consequent increase in intracellular calcium. I also describe for the first time the properties of native TRPM5 channels, including ion selectivity and Ca2+ sensitivity.; These results provide important information for understanding the gating of TRP channels and provide insights into their contribution to physiological processes such as taste.|
|Keyword||TRP channel; taste; PIP2|
|Part of collection||University of Southern California dissertations and theses|
|Publisher (of the original version)||University of Southern California|
|Place of publication (of the original version)||Los Angeles, California|
|Publisher (of the digital version)||University of Southern California. Libraries|
|Legacy record ID||usctheses-m863|
|Repository name||Libraries, University of Southern California|
|Repository address||Los Angeles, California|
|Full text||GATING MECHANISMS OF TRANSIENT RECEPTOR POTENTIAL ION CHANNELS TRPM5 and TRPM4 IN NATIVE AND HETEROLOGOUS CELLS by Zheng Zhang A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (NEUROSCIENCE) December 2007 Copyright 2007 Zheng Zhang|