All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-beta-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two alpha-helices and three beta-strands arranged as beta1-alpha1-beta2-beta3-alpha2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form alpha2 of the second CBS domain (CBS2). We demonstrate that interchanging alpha2 between CLH-3a and CLH-3b interchanges their gating properties. The R-helix of ClC proteins forms part of the ion conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the R-helix linker. C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that alpha2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in alpha2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-like gating and extracellular cysteine reactivity. We postulate that the R-helix linker interacts with CBS2 alpha2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-08152010-213719 |
| Date | 13 December 2010 |
| Creators | Dave', Sonya |
| Contributors | Hassane Mchaourab, Danny Winder, Al Beth, Jens Meiler, Jerod Denton |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-08152010-213719/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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