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Mutational analysis demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane currents

Item Type:Article
Title:Mutational analysis demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane currents
Creators Name:Friedrich, T., Breiderhoff, T. and Jentsch, T.J.
Abstract: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.
Keywords:Cell Line, Cell Membrane, Chloride Channels, Complementary DNA, Ion Channel Gating, Site-Directed Mutagenesis, Patch-Clamp Techniques, Recombinant Proteins, Animals, Xenopus
Source:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology
Page Range:896-902
Date:8 January 1999
Official Publication:https://doi.org/10.1074/jbc.274.2.896
PubMed:View item in PubMed

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