Carboxy-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking [Molecular Bases of Disease]

October 9th, 2015 by Stolting, G., Bungert–Plumke, S., Franzen, A., Fahlke, C.

ClC-K chloride channels are crucial for auditory transduction and urine concentration. Mutations in CLCNKB, the gene encoding the renal chloride channel hClC-Kb, cause Bartter syndrome type III, a human genetic condition characterized by polyuria, hypokalemia and alkalosis. In recent years, several Bartter syndromeassociated mutations have been described that result in truncations of the intracellular carboxy-terminus of hClC-Kb. We here used a combination of whole-cell patch clamp, confocal imaging, co-immunoprecipitation and surface biotinylation to study the functional consequences of a frequent CLCNKB mutation that creates a premature stop codon at W610. We found that W610X leaves the association of hClC-Kb and the accessory subunit barttin unaffected, but impairs its regulation by barttin. W610X attenuates hClC-Kb surface membrane insertion. Moreover, W610X results in hClCKb channel opening in the absence of barttin and prevents further barttin-mediated activation. To describe how the carboxyterminus modifies the regulation by barttin we used V166E rClC-K1. V166E rClC-K1 is active without barttin and exhibits prominent, barttin-regulated voltagedependent gating. Electrophysiological characterization of truncated V166E rClC-K1 demonstrated that the distal carboxyterminus is necessary for slow cooperative gating. Since barttin modifies this particular gating process, channels lacking the distal carboxy-terminal domain are no longer regulated by the accessory subunit. Our results demonstrate that the carboxyterminus of hClC-Kb is not part of the binding site for barttin, but functionally modifies the interplay with barttin. The loss-of-activation of truncated hClC-Kb channels in heterologous expression systems fully explains the reduced basolateral chloride conductance in affected kidneys and the clinical symptoms of Bartter syndrome patients.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Carboxy-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking [Molecular Bases of Disease]