Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel.
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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 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.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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Affiliation: Instituto de Física, Universidad Autonóma de San Luis Potosí, San Luis Potosí, SLP 78290, México.
ABSTRACT
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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. Related in: MedlinePlus |
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Mentions: In a double barrel pore controlled by protopore and common gates, ion conduction occurs when the two gates are open. Fig. 1 (D and E) shows that for mClC-2, the ICl “on” kinetic is dominated by the fast component. This can happen if the slowest (common) gate is partially open and the faster (protopore) gate switches from closed to open. In addition, Fig. 1 C shows that the apparent P0 decreased to zero at positive Vm. This can take place when P0 of the protopore gate (Pf) goes back to approximately zero, assuming that P0 of the common gate (Ps) is >0. Alternative possibilities (Ps = 0 and Pf > 0 or Ps = 0 and Pf = 0) predict slow kinetics, however, we show the presence of a rapid activation. Thus, it seems reasonable to assume that P0 for the common gate would be >0 at positive Vm. An estimation of Ps at positive Vm can be computed assuming that the two gates switch between one open and one closed state, with a fast time constant much faster than the slow time constant and Pf = 0. Under these conditions, ICl will be given by (Bennetts et al., 2001) (4)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{gather*}{\mathrm{I}}_{{\mathrm{Cl}}} \left \left({\mathrm{t}}\right) \right = \\{\mathrm{2Ni}} \left \left[{\mathrm{P}}_{{\mathrm{S{\infty}}}}{\mathrm{P}}_{{\mathrm{f{\infty}}}}-{\mathrm{P}}_{{\mathrm{S0}}} \left \left({\mathrm{P}}_{{\mathrm{f{\infty}}}}-{\mathrm{P}}_{{\mathrm{f0}}}\right) \right {\mathrm{e}}^{{\mathrm{-}}{{\mathrm{t}}}/{{\mathrm{{\tau}f}}}}-{\mathrm{P}}_{{\mathrm{f{\infty}}}} \left \left({\mathrm{P}}_{{\mathrm{S{\infty}}}}-{\mathrm{P}}_{{\mathrm{S0}}}\right) \right {\mathrm{e}}^{{\mathrm{-}}{{\mathrm{t}}}/{{\mathrm{{\tau}s}}}}\right] \right {\mathrm{,}}\end{gather*}\end{document}where N is the number of channels; i is the single channel current; τf and τs are time constants for protopore and common gates, respectively; Ps0 and Ps∞ are the common gate P0 at t = 0 and t = ∞, respectively; and Pf0 and Pf∞ are the protopore gate P0 at t = 0 and t = ∞, respectively. It can be seen from Eq. 4 that ICl would increase rapidly if the common gate is partially open at the holding potential, that is Ps0 > 0. Eq. 4 is similar to Eq. 2, with Af = Ps0/Ps∞ and As = 1 − Ps0/Ps∞. Since at −200 mV, apparent P0 is saturated (Fig. 1), this hints that both Ps and Pf have reached their maximum values, Ps∞ = 1 and Pf∞ = 1, respectively. Therefore, the common gate P0 at the beginning of the −200 mV step would be Ps0 ≈ Af ≈ 0.6 (see Fig. 1 E). These computations indicate that the common gate must be partially open at positive Vm and that the gating of mClC-2 is compatible with the double barrel model controlled by two protopore gates and one common gate whose transitions follow Scheme 1. |
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Affiliation: Instituto de Física, Universidad Autonóma de San Luis Potosí, San Luis Potosí, SLP 78290, México.