Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C122
Functional analysis of novel CLCNKB mutations in patients with Bartter syndrome
M. Keck1, S. L'Hoste1, M. Genete1, M. El Hage1, T. Grand1, R. Vargas-Poussou2, A. Blanchard2, S. Lourdel1, J. Teulon1
1. equipe 3, Centre de recherches des Cordeliers, Paris, France. 2. Genetics, European Georges Pompidou Hospital, Paris, France.
Bartter’s syndrome (BS) is a hereditary salt-wasting tubulopathy characterized by hypokalaemic metabolic alkalosis and secondary hyperaldosteronism (1). BS results from loss-of-function mutations in genes encoding the transport proteins NKCC2, ROMK, ClC-Kb or Barttin (2), which all are involved in NaCl reabsorption in the thick ascending limb of Henle’s loop. Patients with mutations in CLCNKB gene, encoding the ClC-Kb chloride channel, exhibit the most severe hypochloraemic metabolic alkalosis, a phenomenon that could be due to the expression of ClC-Kb in the intercalated cells of the collecting duct (3). To date, mutations in the CLCNKB have been much less investigated than other BS gene mutations. In the present study, we investigated novel CLCNKB mutations collected by the Department of Genetics of the European Georges Pompidou Hospital, including 2 mutations in homozygous patients with BS, (G246R and T659M) and 4 mutations detected in 2 compound heterozygous patients (G120R/W391X and G424R/A204T). The A204T mutation has already been published (4). We studied the wild-type and ClC-Kb mutants in Xenopus laevis oocytes using two-electrode voltage-clamp for measuring CLC-Kb currents and chemiluminescence for evaluating surface expression. Sub-cellular localization was investigated in HEK 293 cells using immunocytochemistry. For the two latter purposes, we designed a ClC-Kb construct carrying an extracellular FLAG epitope that does not alter ClC-Kb currents. Results are shown as mean ± SEM. Experiments included at least 12-30 measurements with 3 different batches of oocytes and 3 replicates with HEK 293 cells. Significance was analyzed with one-way ANOVA follows by Holm-Sidak test. P < 0.05 was considered significant. No significant current was recorded with W391X mutant. G120R, A204T, G246R and G424R mutants produced currents significantly different from those in non injected oocytes but lower by ≈70% as compared to wild-type (wild-type: 4.05±0.17μA, G120R: 1.47±0.19μA, A204T: 1.73±0,15μA, G246R: 1.17±0.17μA and G424R: 1.14±0.17μA). The last mutation (T659M) had no effect on the current amplitude. Accordingly, surface-labeling analysis of ClC-Kb channel showed that W391X was not addressed to the plasma membrane while the labeling of G120R, A204T, G246R and G424R was decreased by more than 70%. That of T659M was not different from wild-type. These results were confirmed by immunocytochemistry on HEK 293 cells, which indicated a defect in membrane targeting for all mutants except T659M. In conclusion, our results show that four of the novel CLCNKB mutations, G120R, G246R, W391X and G424R, are pathogenic and considerably alter membrane targeting of the channel. In contrast, no functional alteration was detected for the T659M mutation, possibly suggesting that it is not directly linked to the disease.
Where applicable, experiments conform with Society ethical requirements