Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel.
(2005)
Journal - The Journal of general physiology (United States )
Abstract :
Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (V(m)) and time dependence of the protopore (P(f)) and common (P(s)) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (I(Cl)) were measured using whole cell patch clamp. WT channels had I(Cl) that showed inward rectification and biexponential time course. Time constants of fast and slow components were approximately 10-fold different at negative V(m) and corresponded to P(f) and P(s), respectively. P(f) and P(s) were approximately 1 at -200 mV, while at V(m) > or = 0 mV, P(f) approximately 0 and P(s) approximately 0.6. Hence, P(f) dominated open kinetics at moderately negative V(m), while at very negative V(m) both gates contributed to gating. At V(m) > or = 0 mV, mClC-2 closes by shutting off P(f). Three- and two-state models described the open-to-closed transitions of P(f) and P(s), respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels. Based on the time and V(m) dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to P(f), the dominant regulator of gating, while the C258 residue alters the V(m) dependence of P(f), probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.
| ISSN : | 0022-1295 |
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| Mesh Heading : | Animals Chloride Channels Chlorides Electrophysiology Ion Channel Gating Kinetics Mice Parotid Gland physiology physiology |
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| Mesh Heading Relevant : | physiology physiology |
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Quantitative Analysis of the Voltage-dependent Gating of Mouse Parotid ClC-2 Chloride Channel
(2005)
Journal - The Journal of General Physiology
Abstract :
Various ClC-type voltage-gated chloride channel isoforms displaya double barrel topology, and their gating mechanisms are thoughtto be similar. However, we demonstrate in this work that thenearly ubiquitous ClC-2 shows significant differences in gatingwhen compared with ClC-0 and ClC-1. To delineate the gatingof ClC-2 in quantitative terms, we have determined the voltage(Vm) and time dependence of the protopore (Pf) and common (Ps)gates that control the opening and closing of the double barrel.mClC-2 was cloned from mouse salivary glands, expressed in HEK293 cells, and the resulting chloride currents (ICl) were measuredusing whole cell patch clamp. WT channels had ICl that showedinward rectification and biexponential time course. Time constantsof fast and slow components were 10-fold different at negativeVm and corresponded to Pf and Ps, respectively. Pf and Ps were1 at –200 mV, while at Vm 0 mV, Pf 0 and Ps 0.6. Hence,Pf dominated open kinetics at moderately negative Vm, whileat very negative Vm both gates contributed to gating. At Vm 0 mV, mClC-2 closes by shutting off Pf. Three- and two-statemodels described the open-to-closed transitions of Pf and Ps,respectively. To test these models, we mutated conserved residuesthat had been previously shown to eliminate or alter Pf or Psin other ClC channels. Based on the time and Vm dependence ofthe two gates in WT and mutant channels, we constructed a modelto explain the gating of mClC-2. In this model the E213 residuecontributes to Pf, the dominant regulator of gating, while theC258 residue alters the Vm dependence of Pf, probably by interactingwith residue E213. These data provide a new perspective on ClC-2gating, suggesting that the protopore gate contributes to bothfast and slow gating and that gating relies strongly on theE213 residue.