Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC67
Novel mutations of CLCNKB gene in patients with Bartter syndrome: a functional analysis in Xenopus laevis oocytes
M. Keck1, S. L'Hoste1, M. Genete1, T. Grand1, R. Vargas-Poussou2, A. Blanchard2, S. Lourdel1, J. Teulon1
1. centre de recherches des Cordeliers, Universite Pierre et Marie Curie, UPMC, Paris, France. 2. Genetics and center for clinical investigation, European Georges Pompidou Hospital, PARIS, France.
Bartter’s syndrome (BS) is an autosomal recessive, 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 (TAL). To date, mutations in the CLCNKB have been much less investigated than other BS gene mutations. Here, we investigated 6 novel (L81P, G246R, R351P, G424R, L439P) and 2 published (A204T, R438H) (3, 4) CLCNKB mutations. The cRNAs for Barttin and FLAG-tagged, wild-type and mutant ClC-Kbs were injected in Xenopus laevis oocytes. CLC-Kb currents were measured using two-electrode voltage-clamp, and surface expression evaluated using a chemiluminescence assay, two-three days after injection. Results are shown as mean +/- SEM. Experiments included at least 12-30 measurements with 3 different batches of oocytes. Significance was analyzed with one-way ANOVA follows by Holm-Sidak test. P < 0.05 was considered significant. No significant current was recorded with G246R, G424R, R438H and L439P mutants. Accordingly, as shown by the chemi-luminescece assay, these mutations were not addressed to the plasma membrane. By contrast, the L81P, A204T and R351P mutants produced currents lower by 40-70% as compared to wild-type ClC-Kb, but significantly different from non injected oocytes (WT: 4.05±0.17μA, L81P: 1.47±0.19μA, A204T: 2.49±0,22μA, R351P: 2.84±0.29μA). Surface expression decreased in similar proportions. Finally, we investigated the effects of changing external Ca2+ from 1 to 10 mM on the currents produced by L81P, A204T and R351P mutants: a 45 to 70% increase was observed indicating that the sensitivity to external calcium,a characteristic property of ClC-Kb /Barttin, was preserved in mutants. In conclusion, all CLCNKB mutations investigated in this study are pathogenic; the functional defects appear to be due to an altered targeting of the channel to the plasma membrane.
Where applicable, experiments conform with Society ethical requirements