a cytoplasmic domain mutation in clc-kb affects long-distance communication across the membrane胞质域突变clc-kb影响跨膜长途通信.pdfVIP

A Cytoplasmic Domain Mutation in ClC-Kb Affects Long- Distance Communication Across the Membrane Gilbert Q. Martinez, Merritt Maduke* Department of Molecular and Cellular Physiology and Program in Biophysics, Stanford University. Stanford, California, United States of America 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