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Characterization of the putative chloride channel xClC‐5 expressed in Xenopus laevis oocytes and comparison with endogenous chloride currents
Author(s) -
Schmieder S.,
Lindenthal S.,
Banderali U.,
Ehrenfeld J.
Publication year - 1998
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1998.379bh.x
Subject(s) - xenopus , chloride channel , chemistry , iodide , biophysics , microbiology and biotechnology , biochemistry , biology , gene , inorganic chemistry
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 β‐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 β‐globin (induced by injection of cRNA presenting the non‐coding regions of the Xenopus β‐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 I β‐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 β‐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 [Ca 2+ ] i increase inhibited I native but had no effect on I β‐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 (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 I β‐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 p K a value (negative log of the acid dissociation constant) of 5.67. 6 In conclusion, I β‐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.