MEMBRANES AND BIOENERGETICS
Mutational Analysis Demonstrates That ClC-4 and ClC-5 Directly Mediate Plasma Membrane Currents*

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ClC-4 and ClC-5, together with ClC-3, form a distinct branch of the CLC chloride channel family. Although ClC-5 was shown to be mainly expressed in endocytotic vesicles, expression of ClC-5 in Xenopus oocytes elicited chloride currents. We now show that ClC-4 also gives rise to strongly outwardly rectifying anion currents when expressed in oocytes. They closely resemble ClC-5 currents with which they share a NO3 > Cl > Br > I conductance sequence that differs from that reported for the highly homologous ClC-3. Both ClC-4 and ClC-5 currents are reduced by lowering extracellular pH. We could measure similar currents after expressing either channel in HEK293 cells. To demonstrate that these currents are directly mediated by the channel proteins, we introduced several point mutations that change channel characteristics. In ClC-5, several point mutations alter the kinetics of activation but leave macroscopic rectification and ion selectivity unchanged. A mutation (N565K) equivalent to a mutation reported to have profound effects on ClC-3 does not have similar effects on ClC-5. Moreover, a mutation at the end of D2 (S168T in ClC-5) changes ion selectivity, and a mutation at the end of D3 (E211A in ClC-5 and E224A in ClC-4) changes voltage dependence and ion selectivity. This shows that ClC-4 and ClC-5 can directly mediate plasma membrane currents.

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This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.