Disease-Causing Dysfunctions of Barttin in Bartter Syndrome Type IV

Abstract

Bartter syndrome type IV is an inherited human condition characterized by severe renal salt wasting and sensorineural deafness. The causal gene, BSND, encodes barttin, an accessory subunit of chloride channels located in the kidney and inner ear. Barttin modulates the stability, cell surface localization, and function of ClC-K channels; distinct mutations cause phenotypes of varying severity. For definition of the molecular basis of this diversity, the functional consequences of six disease-causing mutations (R8L, R8W, G10S, Q32X, G47R, and E88X) on ClC-K channel properties were studied by heterologous expression in renal cell lines, electrophysiology, confocal imaging, and biochemical analysis. Three missense mutations (R8L, R8W, and G10S) eliminated the function of ClC-K/barttin channels but did not prevent the insertion of the channels into the surface membrane. Another mutant that produces a mild renal phenotype (G47R) was capable of performing all functions of wild-type barttin but bound to ClC-K channels less effectively. The nonsense mutation E88X affected epithelial sorting, leading to equal amounts of barttin inserting into the basolateral and apical membranes, contrasting with the preferential apical insertion of wild-type barttin. Last, the nonsense mutation Q32X allowed barttin to associate with ClC-K channels but prevented surface membrane insertion and channel activation. These results demonstrate that Bartter syndrome type IV can be caused by various derangements in the function of barttin, likely contributing to the diversity of observed phenotypes.

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

“…To study the effects of CLC-K truncations on channel expression, intracellular trafficking or function we used heterologous expression in two different cell lines, MDCKII and HEK293T cells (6,26,31). CLC-K/barttin function and subcellular distribution are comparable in both systems (6,31).…”

“…Robust expression and negligible background currents make HEK293T perfectly suited for functional characterization of WT and mutant CLC-K/barttin channels. For studies of channel localization and trafficking we used confluent MDCK II cells that are known to show epithelial properties such as cell polarization and proper sorting and trafficking (26,32) while retaining the electrophysiological signature of ClC-K/barttin in HEK293T cells (31).…”

Hetero-oligomerization of Neuronal Glutamate Transporters

“…Surface Biotinylation and Western Blotting-Cell surface expression of hEAAT3 was assayed with a modification of cell surface biotinylation methods described previously (27,28). In these experiments, hEAAT3 was expressed as a GFP fusion protein because the fluorescence scanner (Typhoon) had preferable imaging capabilities for GFP.…”

Barttin Activates ClC-K Channel Function by Modulating Gating

“…13 In contrast, a BSND mutation that affects only the chaperone function of barttin and leaves the function of ClC-K/barttin channels unaffected caused nonsyndromic deafness. 14 To further understand how barttin modifies the function of ClC-K channels, we studied voltage-dependent gating of two ClC-K isoforms, rat ClC-K1 and human ClC-Ka, expressed either alone or together with barttin.…”