Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel Article uri icon

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 (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 HEK 293 cells, and the resulting chloride currents (ICl) were measured using whole cell patch clamp. WT channels had ICl that showed inward rectification and biexponential time course. Time constants of fast and slow components were ∼10-fold different at negative Vm and corresponded to Pf and Ps, respectively. Pf and Ps were ∼1 at -200 mV, while at Vm ≥ 0 mV, P f ∼ 0 and Ps ∼ 0.6. Hence, Pf dominated open kinetics at moderately negative Vm, while at very negative Vm both gates contributed to gating. At Vm ≥ 0 mV, mClC-2 closes by shutting off Pf. Three- and two-state models described the open-to-closed transitions of Pf and Ps, respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter Pf or Ps in other ClC channels. Based on the time and Vm 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 Pf, the dominant regulator of gating, while the C258 residue alters the Vm dependence of Pf, 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. © The Rockefeller University Press.

publication date

  • 2005-01-01