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Mutational analysis demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane currents
T Friedrich1, T Breiderhoff, T J Jentsch
1Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Hamburg University, Martinistrabetae 52, D-20246, Hamburg, Germany.
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.
Insights
Chloride channel ClC-4 and ClC-5, distinct members of the CLC family, mediate anion currents. Mutations reveal their direct role in plasma membrane currents, differing from ClC-3.
Area of Science:
- Molecular Biology
- Ion Channel Physiology
- Cell Biology
Background:
- ClC-4 and ClC-5 belong to a distinct branch of the CLC chloride channel family.
- While ClC-5 is primarily found in endocytotic vesicles, it elicits chloride currents when expressed in oocytes.
- ClC-3, ClC-4, and ClC-5 share homology but exhibit distinct functional characteristics.
Purpose of the Study:
- To investigate the functional properties of ClC-4 and ClC-5 chloride channels.
- To determine if ClC-4 and ClC-5 directly mediate plasma membrane currents.
- To explore the effects of specific mutations on the function and ion selectivity of ClC-4 and ClC-5.
Main Methods:
- Expression of ClC-4 and ClC-5 in Xenopus oocytes and HEK293 cells.
- Electrophysiological recordings to measure chloride currents.
- Introduction of point mutations to alter channel characteristics and assess functional impact.
Main Results:
- ClC-4 and ClC-5 expression in oocytes produced outwardly rectifying anion currents with a specific ion selectivity (NO3- > Cl- > Br- > I-).
- These currents were reduced by lower extracellular pH and observed in HEK293 cells.
- Point mutations altered channel kinetics, voltage dependence, and ion selectivity, confirming direct channel mediation of currents.
Conclusions:
- ClC-4 and ClC-5 directly mediate plasma membrane anion currents.
- Functional properties and ion selectivity of ClC-4 and ClC-5 differ from the highly homologous ClC-3.
- Specific mutations provide insights into the structure-function relationships of ClC-4 and ClC-5 channels.