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Pathological missense mutations in CLCNKB gene give a wide spectrum of clinical phenotypes in Bartter syndrome type III patients. Molecular analysis of the mutated ClC-Kb channels can be helpful to classify the mutations according to their functional alteration. We investigated the functional consequences of nine mutations in the CLCNKB gene causing Bartter syndrome. We first established that all tested mutations lead to decreased ClC-Kb currents. Combining electrophysiological and biochemical methods in Xenopus laevis oocytes and in MDCKII cells, we identified three classes of mutations. One class is characterized by altered channel trafficking. p.A210V, p.P216L, p.G424R, and p.G437R are totally or partially retained in the endoplasmic reticulum. p.S218N is characterized by reduced channel insertion at the plasma membrane and altered pH-sensitivity; thus, it falls in the second class of mutations. Finally, we found a novel class of functionally inactivated mutants normally present at the plasma membrane. Indeed, we found that p.A204T alters the pH-sensitivity, p.A254V abolishes the calcium-sensitivity. p.G219C and p.G465R are probably partially inactive at the plasma membrane. In conclusion, most pathogenic mutants accumulate partly or totally in intracellular compartments, but some mutants are normally present at the membrane surface and simultaneously show a large range of altered channel gating properties., (© 2019 Wiley Periodicals, Inc.)

Molecules correcting the trafficking (correctors) and gating defects (potentiators) of the cystic fibrosis causing mutation c.1521_1523delCTT (p.Phe508del) begin to be a useful treatment for CF patients bearing p.Phe508del. This mutation has been identified in different genetic contexts, alone or in combination with variants in cis. Until now, 21 exonic variants in cis of p.Phe508del have been identified, albeit at a low frequency. The aim of this study was to evaluate their impact on the efficacy of CFTR-directed corrector/potentiator therapy (Orkambi). The analysis by minigene showed that two out of 15 cis variants tested increased exon skipping (c.609C > T and c.2770G > A). Four cis variants were studied functionally in the absence of p.Phe508del, one of which was found to be deleterious for protein maturation c.1399C > T (p.Leu467Phe). In the presence of p.Phe508del, this variant was the only to prevent the response to Orkambi treatment. This study showed that some patients carrying p.Phe508del complex alleles are predicted to poorly respond to corrector/potentiator treatments. Our results underline the importance to validate treatment efficacy in the context of complex alleles., (© 2017 Wiley Periodicals, Inc.)