The opening and closing of chloride (Cl−) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as “fast gating” is regulated by both external and internal Cl− ions. Although the external Cl− effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl− regulation remains to be characterized. In this study, we examine the internal Cl− effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising Cl−i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of 1 M, a value very different from the one observed in the external Cl− regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties—the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of Cl−i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl− on the fast gating may also be due to the competition of Cl− with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl−. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of Cl−i and charges in the inner pore region. The very different nature of external and internal Cl− regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.
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