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¹ Instituto de Física, Universidad Autonóma de San Luis Potosí, San Luis Potosí, SLP 78290, México
² Department of Medicine, Center for Oral Biology, University of Rochester, Rochester, NY 14642
³ Department of Pharmacology and Physiology, Center for Oral Biology, University of Rochester, Rochester, NY 14642

Correspondence to Jorge Arreola: Arreola{at}dec1.ifisica.uaslp.mx

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 10-fold different at negative V_m and corresponded to P_f and P_s, respectively. P_f and P_s were 1 at –200 mV, while at V_m 0 mV, P_f 0 and P_s 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 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.

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