title: On the mechanism of gating charge movement of ClC-5, a human Cl(-)/H(+) antiporter.
abstract: ClC-5 is a Cl(-)/H(+) antiporter that functions in endosomes and is important for endocytosis in the proximal tubule. The mechanism of transport coupling and voltage dependence in ClC-5 is unclear. Recently, a transport-deficient ClC-5 mutant (E268A) was shown to exhibit transient capacitive currents. Here, we studied the external and internal Cl(-) and pH dependence of the currents of E268A. Transient currents were almost completely independent of the intracellular pH. Even though the transient currents are modulated by extracellular pH, we could exclude that they are generated by proton-binding/unbinding reactions. In contrast, the charge movement showed a nontrivial dependence on external chloride, strongly supporting a model in which the movement of an intrinsic gating charge is followed by the voltage-dependent low-affinity binding of extracellular chloride ions. Mutation of the external Glu-211 (a residue implicated in the coupling of Cl(-) and proton transport) to aspartate abolished steady-state transport, but revealed transient currents that were shifted by ~150 mV to negative voltages compared to E268A. This identifies Glu(ext) as a major component of the gating charge underlying the transient currents of the electrogenic ClC-5 transporter. The molecular events underlying the transient currents of ClC-5 emerging from these results can be explained by an inward movement of the side chain of Glu(ext), followed by the binding of extracellular Cl(-) ions.
authors: Istituto di Biofisica, The National Research Council, Genoa, Italy.
authors: Giovanni
authors: G
authors: Zifarelli
authors: Silvia
authors: S
authors: De Stefano
authors: Ilaria
authors: I
authors: Zanardi
authors: Michael
authors: M
authors: Pusch
mesh_terms: D017920:Antiporters / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology*; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D002713:Chlorine / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D055672:Static Electricity
chemical_list: D017920:Antiporters; D018118:Chloride Channels; D002713:Chlorine; D006859:Hydrogen