Voltage-Dependent Gating of ClC-2 Chloride Channel

The gating of ClC-2 Cl- channel is facilitated by elevated [H+]e and [Cl-]i by interacting with the protopore gate whilst [Cl-]e had not effect. In contrast, the gating of ClC-0 Cl- channel is facilitated by protonation of the protopore gate by intracellular [H+]i in a manner that is dependent on the extracellular [Cl-]e. To gain insights into the Vm dependent mechanism of ClC-2 expressed in HEK cells, we determine the Vm-dependence of open probability (PA(Vm)) at different [H+]i, [H+]e and [Cl-]i using the patch clamp technique. Changing [H+]i by 5 orders of magnitude whilst [Cl-]i/[Cl-]e=140/140 or 10/140 mM did not altered the onset kinetics but channel closing became faster at acidic pHi and PA(Vm) curves were shifted towards more negative Vm. These results suggest that [H+]i did not facilitated gating. In contrast, a same change in [H+]o with [Cl-]i/[Cl-]e=140/140 mM enhanced PA in a bi-phasic manner and shifted PA(Vm) curves to positive Vm. Importantly, PA was >0 with [H+]o=10−10 M and channel closed more slowly when [H+]o or [Cl-]i increased. This implied that ClC-2 can be gated without protonation and that external H+ and/or internal Cl- stabilized the open state. A kinetic analysis of Cl- currents and PA(Vm) curves at different [H+]o and [Cl-]i using a gating scheme coupled to Cl- permeation indicated that protonation of the protopore gate has negligible Vm- and Cl--dependence and that the rate constant for closed-open transition of un-protonated channels were facilitated by elevated [Cl-]i in a Vm-dependent manner. We propose that the majority of the Vm-dependence is due to a Vm-dependent occupancy of ClC-2 pore by the permeant Cl- and that the open conformation is stabilized by a Vm-independent protonation and the Cl- occupancy. Supported by CONACyT.