Sequential interaction of chloride and proton ions with the fast gate steer the voltage-dependent gating in ClC-2 chloride channels

The interaction of either H+ or Cl- ions with the fast gate is the major source of voltage (Vm) dependence in ClC Cl- channels. However, the mechanism by which these ions confer Vm dependence to the ClC-2 Cl- channel remains unclear. By determining the Vm dependence of normalized conductance (Gnorm(Vm)), an index of open probability, ClC-2 gating was studied at different [H+]i, [H+]o and [Cl-]i. Changing [H+]i by five orders of magnitude whilst [Cl-]i/[Cl-]o= 140/140 or 10/140 mm slightly shifted Gnorm(Vm) to negative Vm without altering the onset kinetics; however, channel closing was slower at acidic pHi. A similar change in [H+]o with [Cl-]i/[Cl-]o= 140/140 mm enhanced Gnorm in a bell-shaped manner and shifted Gnorm(Vm) curves to positive Vm. Importantly, Gnorm was >0 with [H+]o= 10-10 m but channel closing was slower when [H+]o or [Cl-]i increased implying that ClC-2 was opened without protonation and that external H+ and/or internal Cl- ions stabilized the open conformation. The analysis of kinetics and steady-state properties at different [H+]o and [Cl-]i was carried out using a gating Scheme coupled to Cl- permeation. Unlike previous results showing Vm-dependent protonation, our analysis revealed that fast gate protonation was Vm and Cl- independent and the equilibrium constant for closedopen transition of unprotonated channels was facilitated by elevated [Cl-]i in a Vm-dependent manner. Hence a Vm dependence of pore occupancy by Cl- induces a conformational change in unprotonated closed channels, before the pore opens, and the open conformation is stabilized by Cl- occupancy and Vm-independent protonation.