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

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Abstract
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 Xenopusβ‐globin sequence and in which a Kozak consensus site was introduced before the initiator ATG. We then compared the induced currents Inative (induced by injection of cRNA presenting the native non‐coding regions of xClC‐5) and Iβ‐globin (induced by injection of cRNA presenting the non‐coding regions of the Xenopusβ‐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 (β) xClC‐5 cRNA, (2) the Iβ‐globin outward rectification score was 2.6 times that of Inative, (3) the anion conductivity sequence was nitrate > bromide > chloride > iodide >> gluconate for Iβ‐globin and iodide > bromide > nitrate > chloride >> gluconate for Inative, (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 Inative but had no effect on Iβ‐globin, and (5) Inative showed considerable similarity to the previously reported endogenous current appearing after ClC‐6 or pICln cRNA injection. Comparison of Inative with the endogenous chloride current ICl,swell which develops under hyposmotic conditions demonstrated several similarities in their electrophysiological and pharmacological characteristics but were nevertheless distinguishable. In vitro translation assays demonstrated that protein synthesis was much greater using the (β) xClC‐5 construct than that of xClC‐5. Furthermore, immunoreactivity of membrane preparations of Xenopus oocytes was only observed with the (β) xClC‐5 construct, its intensity being positively correlated with Iβ‐globin levels. In addition, the current induced in (β) xClC‐5 cRNA‐injected oocytes presented a very marked pH dependence (inhibition by acid external media) with a pKa value (negative log of the acid dissociation constant) of 5.67. In conclusion, Iβ‐globin may be due to the presence of xClC‐5 in the oocyte plasma membrane playing a role as an anion channel whereas Inative 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.