Abstract | BACKGROUND: ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix. METHODOLOGY/PRINCIPAL FINDINGS: Two- electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface. CONCLUSIONS: The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change--ranging from the cytoplasmic side of the protein to the extracellular side of the protein--is involved in the Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure.
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Authors | Gilbert Q Martinez, Merritt Maduke |
Journal | PloS one
(PLoS One)
Vol. 3
Issue 7
Pg. e2746
(Jul 23 2008)
ISSN: 1932-6203 [Electronic] United States |
PMID | 18648499
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- CLCNKA protein, human
- CLCNKB protein, human
- Chloride Channels
- Calcium
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Topics |
- Animals
- Bartter Syndrome
(diagnosis, genetics)
- Calcium
(metabolism)
- Chloride Channels
(genetics, physiology)
- Cytoplasm
(metabolism)
- Ear, Inner
(metabolism)
- Electrophysiology
- Humans
- Kidney
(metabolism)
- Mutation
- Oocytes
(metabolism)
- Protein Structure, Quaternary
- Protein Structure, Tertiary
- Xenopus
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