title: Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking.
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.
authors.affiliation: From the Institute of Complex Systems 4 (ICS-4), Zelluläre Biophysik, Forschungszentrum Jülich, 52425 Jülich g.stoelting@fz-juelich.de.
authors.forename: Gabriel
authors.identifier: http://orcid.org/0000-0002-2339-0545
authors.initials: G
authors.lastname: Stölting
authors.affiliation: From the Institute of Complex Systems 4 (ICS-4), Zelluläre Biophysik, Forschungszentrum Jülich, 52425 Jülich.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Bungert-Plümke
authors.affiliation: From the Institute of Complex Systems 4 (ICS-4), Zelluläre Biophysik, Forschungszentrum Jülich, 52425 Jülich.
authors.forename: Arne
authors.initials: A
authors.lastname: Franzen
authors.affiliation: From the Institute of Complex Systems 4 (ICS-4), Zelluläre Biophysik, Forschungszentrum Jülich, 52425 Jülich c.fahlke@fz-juelich.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Fahlke
mesh_terms: D000818:Animals; D001665:Binding Sites; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D018389:Codon, Nonsense / Q000235:genetics; D004285:Dogs; D057809:HEK293 Cells; D006801:Humans; D007008:Hypokalemia / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D007668:Kidney / Q000378:metabolism / Q000473:pathology; D061985:Madin Darby Canine Kidney Cells; D011141:Polyuria / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D021381:Protein Transport / Q000235:genetics
chemical_list: C440005:BSND protein, human; C412733:CLCNKB protein, human; D018118:Chloride Channels; D018389:Codon, Nonsense
keywords: Bartter syndrome; accessory subunits; chloride channel; epithelial transport; gating; intracellular trafficking; patch clamp; renal physiology