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Characterization of the putative chloride channel xClC-5 expressed in Xenopus laevis oocytes and comparison with endogenous chloride currents

S. Schmieder, S. Lindenthal, U. Banderali and J. Ehrenfeld

1. We recently cloned a putative chloride channel (xClC-5) from the renal cell line A6, which induced the appearance of a Cl^- conductance not found in control oocytes after homologous expression in Xenopus oocytes. With the aim of increasing the Xenopus oocyte xClC-5 expression, we constructed a new plasmid in which the native 5' and 3' non-coding regions of xClC-5 were replaced by the non-coding regions of the Xenopus -globin sequence and in which a Kozak consensus site was introduced before the initiator ATG.

2. We then compared the induced currents I_native (induced by injection of cRNA presenting the native non-coding regions of xClC-5) and I_-globin (induced by injection of cRNA presenting the non-coding regions of the Xenopus -globin sequence) investigating anion selectivity and anion blocker sensitivity. Several differences were found: (1) expression yield and oocyte surviving rate were largely increased by injecting () xClC-5 cRNA, (2) the I_-globin outward rectification score was 2·6 times that of I_native, (3) the anion conductivity sequence was nitrate > bromide > chloride > iodide >> gluconate for I_-globin and iodide > bromide > nitrate > chloride >> gluconate for I_native, (4) 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), anthracene-9-carboxylic acid (9-AC), DIDS, lanthanum ions, cAMP and ionomycin-induced [Ca²⁺]_i increase inhibited I_native but had no effect on I_-globin, and (5) I_native showed considerable similarity to the previously reported endogenous current appearing after ClC-6 or pICln cRNA injection.

3. Comparison of I_native with the endogenous chloride current I_Cl,swell which develops under hyposmotic conditions demonstrated several similarities in their electrophysiological and pharmacological characteristics but were nevertheless distinguishable.

4. In vitro translation assays demonstrated that protein synthesis was much greater using the () xClC-5 construct than that of xClC-5. Furthermore, immunoreactivity of membrane preparations of Xenopus oocytes was only observed with the () xClC-5 construct, its intensity being positively correlated with I_-globin levels.

5. In addition, the current induced in () xClC-5 cRNA-injected oocytes presented a very marked pH dependence (inhibition by acid external media) with a pK_a value (negative log of the acid dissociation constant) of 5·67.

6. In conclusion, I_-globin may be due to the presence of xClC-5 in the oocyte plasma membrane playing a role as an anion channel whereas I_native may represent an endogenous current induced by xClC-5 cRNA injection. The use of antibodies will facilitate the tissue and subcellular localization of xClC-5 and the identification of its physiological role.

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