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

ClC-4 and ClC-5, together with ClC-3, form a distinct branch of the CLC chl oride channel family. Although ClC-5 was shown to be mainly expressed in en docytotic vesicles, expression of ClC-5 in Xenopus oocytes elicited chlorid e currents. We now show that ClC-4 also gives rise to strongly outwardly re ctifying anion currents when expressed in oocytes. They closely resemble Cl C-5 currents with which they share a NO3- > Cl- > Br- > I- conductance sequ ence 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. T o demonstrate that these currents are directly mediated by the channel prot eins, we introduced several point mutations that change channel characteris tics. In ClC-5, several point mutations alter the kinetics of activation bu t 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 o f 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 me mbrane currents.