Electrostatic control and chloride regulation of the fast gating of ClC-0 chloride channels.
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.
Center for Neuroscience University of California-Davis, 1544 Newton Court, Davis, CA 95616, USA. tycchen@ucdavis.edu
Tsung-Yu
TY
Chen
Mei-Fang
MF
Chen
Chia-Wei
CW
Lin
D000818:Animals; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D002712:Chlorides / Q000494:pharmacology / Q000502:physiology; D005260:Female; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D055672:Static Electricity; D014981:Xenopus
C120501:CLC-0 chloride channel protein, Torpedo californica; D018118:Chloride Channels; D002712:Chlorides