Analysis ofCLCNKBmutations at dimer‐interface, calcium‐binding site, and pore reveals a variety of functional alterations in ClC‐Kb channel leading to Bartter syndrome

Abstract

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.

A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl–/H+ transport cycle

“…Thus, our data suggest that the Helix-O motions involved in transition to the outward-facing state are distinct from those postulated to facilitate inner-gate opening. While the mechanistic details linking Helix O-Q movements between outward-and inwardfacing states are currently unknown, it is of interest to note that many disease-causing mutations occur in this segment, in both CLC channels (Saviane et al, 1999;Pusch, 2002;Bignon et al, 2020) and transporters (Lourdel et al, 2012;Veeramah et al, 2013). In Helix O, for example, mutation of a highly conserved glycine residue occurring mid-helix can cause Dent's disease (CLC-5, (Smith et al, 2009)) or Bartter syndrome (CLC -Kb, (Lin et al, 2009)).…”

Genetic diagnosis of Bartter syndrome in Iranian patients and detection of a novel homozygous CLCNKB mutation

“…20 In addition, tow cystathionineβsynthase domains located at the end of the carboxyl terminus have important roles in the function and/or expression of CLC-Kb, and eliminating these domains due to the mutation may abolish their functions. 1,20,21 It is noteworthy that this deletion has been previously reported in the compound heterozygote state. 22 This is the first time this mutation c.1332_1335delCTCT is reported in the homozygous state.…”

“…CLC‐Ka and CLC‐Kb belong to a large CLC gene family of chloride channels and are the most voltage‐gated Cl − channels located in the kidney and the inner ear cells. 1 , 8 CLC‐Kb operates as a homodimer associated with the regulatory Barttin subunit leading to reabsorption of Cl − at thick ascending limb (TAL) of loop of Henle. 8 , 9 This transmembrane protein is encoded by Cl − voltage‐gated channel Kb ( CLCNKB ) gene [OMIM #602023] which is situated on chromosome 1p36 and includes 20 exons.…”

“…CLC‐Ka and CLC‐Kb belong to a large CLC gene family of chloride channels and are the most voltage‐gated Cl − channels located in the kidney and the inner ear cells 1,8 . CLC‐Kb operates as a homodimer associated with the regulatory Barttin subunit leading to reabsorption of Cl − at thick ascending limb (TAL) of loop of Henle 8,9 .…”

“…

Bartter syndrome, BS, is a rare disease with a prevalence of 1 in 1,000,000 and a heterogeneous group of salt-wasting tubulopathies distinguished by polyuria, polydipsia, hypokalemia, metabolic alkalosis, renal salt loss with normal or low blood pressure and secondary hyperaldosteronism. [1][2][3] Genotypically, BS divided into at least 6 types results from loss of function mutations in different genes encoding the transporters involved in salt reabsorption at the TAL. [3][4][5] The type III BS appears in infancy to adolescence and has a wide spectrum of phenotype variability with a predominantly mild presentation.

…”