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.

The SLC26 gene family encodes multifunctional transport proteins in numerous tissues and organs. Some paralogs function as anion exchangers, others as anion channels, and one, prestin (SLC26A5), represents a membrane-bound motor protein in outer hair cells of the inner ear. At present, little is known about the molecular basis of this functional diversity. We studied the subunit stoichiometry of one bacterial, one teleost, and two mammalian SLC26 isoforms expressed in Xenopus laevis oocytes or in mammalian cells using blue native PAGE and chemical cross-linking. All tested SLC26s are assembled as dimers composed of two identical subunits.Co-expressionoftwomutantprestinswithdistinctvoltagedependent capacitances results in motor proteins with novel electrical properties, indicating that the two subunits do not function independently. Our results indicate that an evolutionarily conserved dimeric quaternary structure represents the native and functional state of SLC26 transporters.

In an effort to gain further insight into the conformational and topographical requirements for recognition by the N-terminal SH2 domain of protein tyrosine phosphatase SHP-1, we synthesized a series of linear and cyclic peptides derived from the sequence surrounding phosphotyrosine 2267 in the receptor tyrosine kinase Ros (EGLNpYMVL). A molecular modeling approach was used to suggest peptide modifications sterically compatible with the N-SH2-peptide binding groove and possibly enhanced binding affinities compared to the parent peptide. The potencies of the synthesized compounds were evaluated by assaying their ability to stimulate phosphatase activity as well as by their binding affinities to the GST-fused N-SH2 domain of SHP-1. In the series of linear peptides, structural modifications of Ros pY2267 in positions pY + 1 to pY + 3 by amino acid residues structurally related to Phe, for example l-erythro/threo-Abu(betaPh) (5a, 5b), yielded ligands with increased binding affinity. The incorporation of d-amino acid residues at pY + 1 and pY + 3 led to inactive peptides. The replacement of Phe in both pY + 1 and pY + 3 by Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was also not tolerated due to steric hindrance. Cyclic peptides (13, 14) that were linked via residues in positions pY - 1 (Lys) and pY + 2 (Asp/Glu) and contained a Gly residue in the bridging unit displayed much lower potencies for the stimulation of SHP-1 activity but increased binding affinities compared to Ros pY2267. They partially competed with Ros pY2267 in the activation assay. Such cyclic structures may serve as scaffolds for competitive SHP-1 inhibitor design targeting N-SH2 domain-protein interactions that block SHP-1 activation.

Inactivation of voltage-gated calcium channels is crucial for the spatiotemporal coordination of calcium signals and prevention of toxic calcium buildup. Only one member of the highly conserved family of calcium channel ␤-subunits-CaV␤-inhibits inactivation. This unique property has been attributed to short variable regions of the protein; however, here we report that this inhibition actually is conferred by a conserved guanylate kinase (GK) domain and, moreover, that this domain alone recapitulates CaV␤-mediated modulation of channel activation. We expressed and refolded the GK domain of CaV␤2a, the unique variant that inhibits inactivation, and of CaV␤1b, an isoform that facilitates it. The refolded domains of both CaV␤ variants were found to inhibit inactivation of CaV2.3 channels expressed in Xenopus laevis oocytes. These findings suggest that the GK domain endows calcium channels with a brake restraining voltage-dependent inactivation, and thus facilitation of inactivation by full-length CaV␤ requires additional structural determinants to antagonize the GK effect. We found that CaV␤ can switch the inactivation phenotype conferred to CaV2.3 from slow to fast after posttranslational modifications during channel biogenesis. Our findings provide a framework within which to understand the modulation of inactivation and a new functional map of CaV␤ in which the GK domain regulates channel gating and the other conserved domain (Src homology 3) may couple calcium channels to other signaling pathways.auxiliary subunit ͉ modular structure ͉ regulation C alcium signals mediate various cellular processes, including neurotransmission, excitation-contraction coupling, hormone secretion, and gene expression (1). Voltage-gated calcium channels (VGCCs) are activated and inactivated on membrane depolarization, allowing transient increases in cytosolic Ca 2ϩ concentration. Voltage-dependent activation and inactivation of VGCCs depend strongly on the association of the ancillary ␤-subunit (Ca V ␤) to a highly conserved sequence within the intracellular loop joining the first and second repeats (loop I-II) of the pore-forming subunit (Ca V ␣ 1 ), known as the ␣-interaction domain (AID) (2). Ca V ␤ is encoded by four nonallelic genes (␤ 1-4 ), each with multiple splice variants. Except for a few short splicing forms (3), all of these genes share a common structural arrangement, consisting of two highly conserved regions separated and flanked by shorter variable sequences (Fig. 1A). Crystallographic studies have revealed that whereas the first region encompasses a Src homology 3 (SH3) domain, the second region encompasses a guanylate kinase (GK) domain. The AID sequence forms an ␣-helix that fits into a hydrophobic cleft of the GK module that lies on the opposite side of the SH3 domain (Fig. 1B) (4-6). This suggests that the isolated GK module may preserve at least some of the modulatory capabilities of the full Ca V ␤.Despite several attempts to use different Ca V ␤ constructs and experimental approaches (7-11), the functional ...