Members of the ubiquitously expressed CLC "Chloride Channel" family of transport proteins facilitate the transport of chloride across cellular membranes. In humans, the striking diversity of diseases that arise from disruption of CLCs underscores their importance in a variety of different cellular processes. Since a number of disease-causing mutations occur in the cytoplasmic domains of the CLCs, I hypothesized that these domains play a role in regulating the permeation of ions across the membrane. To test this hypothesis, I performed electrophysiological experiments on disease-causing mutant channels. While several mutants displayed electrophysiological phenotypes, one of these, the R538P mutant of the kidney CIC-Kb chloride channel, has a particularly remarkable phenotype. Specifically, this mutation disrupts activation of the channel by extracellular calcium. This result shows that there is long distance communication between the cytoplasm and the extracellular domain — across the entire membrane. To elucidate the structural mechanism for this allosteric regulation, I performed a series of biochemical and biophysical experiments using recombinant protein corresponding to the C-terminal cytoplasmic domain of C1C-Kb. These experiments revealed that the structural changes between the wild type and mutant protein are highly localized and that the quaternary structure of the C1C-Kb domain is identical to that observed in other CLC cytoplasmic domains. The CLC cytoplasmic domains contain conserved protein motifs called "CBS Domains", which are present in a large number of disparate protein families. Using computational methods, I demonstrated that the R538P mutation is located on a loop that interacts with the most highly conserved amino acids in the CBS Domains. This result led me to propose a mechanism that could confer communication between the CBSDs and the transmembrane channel. This mechanism may be broadly applicable to the many unrelated proteins that consist of a catalytic domain attached to CBS domains.
|School Location:||United States -- California|
|Source:||DAI-B 69/05, Dissertation Abstracts International|
|Keywords:||Bartter's syndrome, CBS domains, CBSD, CIC-Kb, CLC, Chloride channels, Transmembrane|
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