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. 2009 Jan;20(1):145-53.
doi: 10.1681/ASN.2008010102. Epub 2008 Sep 5.

Disease-causing dysfunctions of barttin in Bartter syndrome type IV

Audrey G H Janssen  1 , Ute Scholl, Constanze Domeyer, Doreen Nothmann, Ariane Leinenweber, Christoph Fahlke
Affiliations

Disease-causing dysfunctions of barttin in Bartter syndrome type IV

Audrey G H Janssen et al. J Am Soc Nephrol. 2009 Jan.

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.

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Figures

Figure 1.
Figure 1.
Disease-causing barttin mutations result in decreased amplitudes of ClC-Ka/barttin and ClC-Kb/barttin anion currents. (A through D) Representative currents from cells expressing ClC-Ka (A and B) or ClC-Kb (C and D) without (A and C) or with (B and D) barttin. (E and F) Current-voltage relationships from cells expressing ClC-Ka (E) or ClC-Kb (F) either with or without barttin (n > 4). (G and H) Mean isochronal current amplitudes determined at voltage steps to −135 mV from cells expressing ClC-Ka (G) or ClC-Kb (H) with various pathogenic barttin mutations (n ≥ 3). **P < 0.01, *P < 0.05 versus WT barttin.
Figure 2.
Figure 2.
Biochemical analysis of barttin and ClC-Kb. (A) Fluorescence scan of an SDS-PAGE gel of CFP-tagged WT and mutant barttin proteins heterologously expressed in tsA201 cells. (B) SDS-PAGE gel (6 to 15% polyacrylamide) showing the effect of Endo-H and PNGase F on YFP-ClC-Kb glycosylation in the presence and absence of WT barttin in MDCK cells. The same gel was scanned at two different intensities to show complex glycosylation (#, top) as well as core-glycosylated (•) or not glycosylated (○) protein. (C) SDS-PAGE gel (6 to 15% polyacrylamide) of YFP-ClC-Kb either transfected alone or co-transfected with WT or mutant barttin in MDCKII cells. (D) Ratio of the intensities of complex glycosylated to total YFP-ClC-Kb fluorescence from cells coexpressing WT or mutant barttin. Data are means ± SEM from five or more experiments. (E) YFP-ClC-Kb fluorescence from cells coexpressing WT or mutant barttin from at least seven experiments. For each experiment, fluorescence was normalized to YFP-ClC-Kb fluorescence in the presence of WT barttin as indicated by the gray line. **P < 0.01, *P < 0.05 versus WT barttin.
Figure 3.
Figure 3.
Subcellular distribution of WT and mutant barttin. (A through G) Fluorescence confocal images of living MDCK cells expressing WT or mutant barttin-CFP fusion proteins. Bar = 5 μm.
Figure 4.
Figure 4.
Effects of disease-causing barttin mutations on the localization of ClC-Kb/barttin channels in living MDCKII cells. (A through G) Representative confocal images of cells expressing YFP-ClC-Kb together with CFP tagged WT (A), R8L (B), R8W (C), G10S (D), Q32X (E), G47R (F), and E88X (G) barttin. CFP is shown in green, and YFP is shown in red. This color code results in an orange coloring of regions where both proteins overlap. Individual YFP and CFP fluorescences are added to demonstrate co-localization of WT barttin and ClC-Kb. Bar = 5 μm. (H) Fluorescence scan of an SDS-PAGE gel of YFP-ClC-Kb present in the total cleared lysate and the purified surface proteins, when expressed alone or in the presence of WT or mutant barttin. (I) Membrane surface insertion of ClC-Kb by mutant barttin was quantified by dividing the fluorescence of surface-biotinylated YFP-ClC-Kb by the fluorescence of the total cleared lysate and normalization to the ratio found in the presence of WT barttin. Data are means ± SEM from three experiments. **P < 0.01.
Figure 5.
Figure 5.
Epithelial sorting of ClC-Kb by WT and mutant barttin. (A and B) Confocal images of polarized MDCK cells stably expressing WT (A) or E88X (B) barttin-CFP. Cells were grown on filters and fixed before imaging. x-y projections (front view) are given in the upper part, x-z projections (side view) in the lower part. Bar = 5μm. (C) Fluorescence scan of an SDS-PAGE of YFP-ClC-Kb purified by surface biotinylation from basolateral and apical membranes of MDCK cells expressing WT or mutant barttin together with YFP-ClC-Kb. (D) Relative amounts of ClC-Kb channels in the apical and the basolateral membrane. YFP-ClC-Kb surface-biotinylated from the apical or the basolateral membrane was quantified by YFP fluorescence, adjusted for actin amounts, and then given as relative amount of the total surface fluorescence. Data are means ± SEM from five experiments. **P < 0.01.
Figure 6.
Figure 6.
Pathogenic missense mutations affect the function of ClC-K/barttin channels. (A through D) Representative current recordings from cells expressing hClC-1 alone (A), ClC-Kb with barttin (B), concatameric hClC-1-ClC-Kb without barttin (C), or together with barttin (D). (E) Voltage dependence of the normalized instantaneous current amplitude from cells expressing hClC-1-ClC-Kb concatamers alone or together with WT or mutant barttin. Data are means ± SEM from between two and seven cells.
Figure 7.
Figure 7.
Modification of V166E rClC-K1 by WT or mutant barttin. (A through D) Representative current recordings from cells expressing V166E rClC-K1 channels, without barttin (A) or with WT (B), Q32X (C), or G47R (D) barttin. (E and F) Time dependences of the mean current amplitudes and variances on the given pulse protocols, and plots of variances versus mean current amplitudes for V166E rClC-K1 channels, with WT (E) or G47R (F) barttin. The solid lines are fits of the function σ2 = iI(t) − I(t)2/N. (G) Voltage dependence of the absolute open probabilities of V166E rClC-K1/mutant barttin channels. Data are means ± SEM from at least seven cells.
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