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  • 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 Xenopusβ-globin sequence and in which a Kozak consensus site was introduced before the initiator ATG.
  • 2
    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.
  • 3
    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.
  • 4
    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.
  • 5
    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.
  • 6
    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.