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Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking.
- Source :
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The Journal of biological chemistry [J Biol Chem] 2015 Dec 18; Vol. 290 (51), pp. 30406-16. Date of Electronic Publication: 2015 Oct 09. - Publication Year :
- 2015
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Abstract
- 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 syndrome-associated mutations have been described that result in truncations of the intracellular carboxyl 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 Trp-610. 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 hClC-Kb channel opening in the absence of barttin and prevents further barttin-mediated activation. To describe how the carboxyl terminus modifies the regulation by barttin we used V166E rClC-K1. V166E rClC-K1 is active without barttin and exhibits prominent, barttin-regulated voltage-dependent gating. Electrophysiological characterization of truncated V166E rClC-K1 demonstrated that the distal carboxyl terminus is necessary for slow cooperative gating. Since barttin modifies this particular gating process, channels lacking the distal carboxyl-terminal domain are no longer regulated by the accessory subunit. Our results demonstrate that the carboxyl terminus 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.<br /> (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Subjects :
- Animals
Binding Sites
Chloride Channels genetics
Codon, Nonsense genetics
Dogs
HEK293 Cells
Humans
Hypokalemia genetics
Hypokalemia metabolism
Kidney metabolism
Kidney pathology
Madin Darby Canine Kidney Cells
Polyuria genetics
Polyuria metabolism
Polyuria pathology
Protein Transport genetics
Chloride Channels metabolism
Ion Channel Gating
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 290
- Issue :
- 51
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 26453302
- Full Text :
- https://doi.org/10.1074/jbc.M115.675827