Key result
Raising internal chloride concentration reduces the closing rate of the ClC-0 fast gate with an apparent affinity of ~1 M, and mutating charged residues in the pore similarly alters closing rates.
Internal chloride concentration and pore charge mutations primarily affect the closing rate of ClC-0 channels, suggesting a nonequilibrium cycle in fast gating.
Supports nonequilibrium fast gating in ClC-0; leaves open relevance to mammalian CLC channels or disease.
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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Chen et al. (2003) studied this question. Internal chloride concentration and pore mutations vs. Wild-type channel / low internal chloride was evaluated on Fast-gate opening and closing rates. Raising internal chloride concentration reduces the closing rate of the ClC-0 fast gate with an apparent affinity of ~1 M, and mutating charged residues in the pore similarly alters closing rates.
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