Abstract
ClC-K channels belong to the CLC family of chloride channels and are predominantly expressed in the kidney. Genetic evidence suggests their involvement in transepithelial transport of chloride in distal nephron segments; ClC-K1 gene deletion leads to nephrogenic diabetes insipidus in mice, and mutations of the hClC-Kb gene cause Bartter's syndrome type III in humans. Expression of rClC-K1 in Xenopus oocytes yielded voltage-independent currents that were pH-sensitive, had a Br(-) > NO(3)(-) = Cl(-) > I(-) conductance sequence, and were activated by extracellular calcium. A glutamate for valine exchange at amino acid position 166 induced strong voltage dependence and altered the conductance sequence of ClC-K1. This demonstrates that rClC-K1 indeed functions as an anion channel. By contrast, we did not detect currents upon hClC-Kb expression in Xenopus oocytes. Using a chimeric approach, we defined a protein domain that, when replaced by that of rClC-K1, allowed the functional expression of a chimera consisting predominantly of hClC-Kb. Its currents were linear and were inhibited by extracellular acidification. Contrasting with rClC-K1, they displayed a Cl(-) > Br(-)> I(-) > NO(3)(-) conductance sequence and were not augmented by extracellular calcium. Insertion of point mutations associated with Bartter's syndrome type III destroyed channel activity. We conclude that ClC-K proteins form constitutively open chloride channels with distinct physiological characteristics.
Highlights
OcClC-Ka) at the basolateral side of the thick ascending limb and other more distally located nephron segments [8, 9]
Genetic evidence suggests their involvement in transepithelial transport of chloride in distal nephron segments; ClC-K1 gene deletion leads to nephrogenic diabetes insipidus in mice, and mutations of the hClC-Kb gene cause Bartter’s syndrome type III in humans
Similar to that study, current amplitudes were rather low (ϳ1 A at ϩ60 mV) and difficult to discriminate from endogenous oocyte chloride currents
Summary
OcClC-Ka) at the basolateral side of the thick ascending limb and other more distally located nephron segments [8, 9]. Mutations in the hClC-Kb gene (CLCNKB) are associated with Bartter’s syndrome [11] This autosomal recessive salt-wasting disorder is characterized by a reduced sodium chloride reabsorption in the thick ascending limb. This puts hClC-Kb into a functional relationship with the apical NaK2Cl cotransporter and with the ROMK potassium channel, whose inactivation causes other variants of Bartter’s syndrome [12, 13]. Uchida et al reported that expression of rat ClC-K1 in Xenopus oocytes induced outwardly rectifying chloride currents that resembled those observed in the ascending thin limb [7] These currents are inhibited by acidic extracellular pH or by reducing the extracellular calcium concentration, and have an anion conductivity sequence of BrϪ Ͼ ClϪ Ͼ IϪ [4]. This suggests that Bartter’s syndrome III is due to the loss of a chloride current with characteristics that differ from those of ClC-K1 currents
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