Assessing CLC-2 Chloride Channel Voltage Gating by Pore Occupation with Acetate

Abstract

The activation of the homodimeric ClC-2 Cl- channel is voltage dependent. Protopore gates control each of the two pores of the ClC-2 channel. The closed to open transition is initiated by dislodging the negatively charged side chain of a glutamate residue, which constitute the protopore gate located inside the pore. We hypothesize that this gating transition is triggered when intracellular anions occupy the pore. Thus, we investigated channel activation using acetate, a low permeant anion (Pace/PCl = 0.04) that does not traverse the pore. The cells were bathed in a solution containing 140 mM Cl- (pH 7.3) and channel activation was evaluated by measuring the tail current at +80 mV after application of test pulses from +40 to −200 mV. When cells were dialyzed with 140 mM acetate there was no activation, perhaps because acetate failed to occupy the pore. To verify this we compared the Vm-dependent activation of ClC-2 in cells dialyzed with acetate + Cl- (80 + 60 mM) versus Cl- alone (60 mM). These experiments showed that channel activation was shifted by +60 mV in cells dialyzed with the acetate + Cl- mixture relative to that obtained with 60 mM Cl-. Next the charge of the acetate was neutralized by lowering the pHi from 7.3 to 4.2. Since this manipulation ablated the positive shift in activation observed with the mixture we concluded that activation was mediated by multi-ion occupancy. Our data were reproduced by a pore model containing two anion binding sites. Thus, we propose that after a hyperpolarization the intracellular anions occupy the pore and force the gate open to allow anion permeation. Supported by grant 219949 from CONACyT.