Cysteine modification of a putative pore residue in ClC-0: implication for the pore stoichiometry of ClC chloride channels.
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The fast gate of the MTSEA-modified K165C homodimer responded to external Cl(-) less effectively, so the P(o)-V curve was shifted to a more depolarized potential by approximately 45 mV.These results showed that K165 is important for both the fast and slow gating of ClC-0.Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate.
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PubMed Central - PubMed
Affiliation: Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.
ABSTRACT
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The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share approximately 50-60% amino acid identity and show similar gating behaviors. Although they both contain two subunits, the number of pores present in the homodimeric channel is controversial. The double-barrel model proposed for ClC-0 was recently challenged by a one-pore model partly based on experiments with ClC-1 exploiting cysteine mutagenesis followed by modification with methanethiosulfonate (MTS) reagents. To investigate the pore stoichiometry of ClC-0 more rigorously, we applied a similar strategy of MTS modification in an inactivation-suppressed mutant (C212S) of ClC-0. Mutation of lysine 165 to cysteine (K165C) rendered the channel nonfunctional, but modification of the introduced cysteine by 2-aminoethyl MTS (MTSEA) recovered functional channels with altered properties of gating-permeation coupling. The fast gate of the MTSEA-modified K165C homodimer responded to external Cl(-) less effectively, so the P(o)-V curve was shifted to a more depolarized potential by approximately 45 mV. The K165C-K165 heterodimer showed double-barrel-like channel activity after MTSEA modification, with the fast-gating behaviors mimicking a combination of those of the mutant and the wild-type pore, as expected for the two-pore model. Without MTSEA modification, the heterodimer showed only one pore, and was easier to inactivate than the two-pore channel. These results showed that K165 is important for both the fast and slow gating of ClC-0. Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate. |
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Mentions: The MTSEA modification rate was examined with pulsing protocol 2 at 0.5 Hz. Because the induced current was measured at +40 mV where the fast gate opens completely (Po ≅ 1) in both the homo- and heterodimers, the apparent current induction rate is not influenced by the fast-gate open probability. On the other hand, the current induction rate is affected by the slow-gate open probability of the channel. Fig. 9 A (below) depicts two schemes for the MTSEA modifications in the homodimeric and heterodimeric channels. In low concentrations of MTSEA, the reaction schemes can be further reduced to linear models with two (S1 and S2 in the heterodimer) and three (D0, D1 and D2 in the homodimer) states, where S and D represent single and double cysteine mutations and the subscripts denote the number of the open pores. Let α and β be the slow-gating transition rates, λ and μ the pseudo-first-order on and off rate for K165C modification, and PS1, PS2, PD1, and PD2 the slow-gate open probabilities of S1, S2, D1, and D2 states, respectively. When μ is small, the normalized current-induction time courses of the heterodimeric and homodimeric channels are approximated by and : 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{equation*}I_{{\mathrm{S}}} \left \left(t\right) \right =1- \left \left(1-{0.5\;P_{{\mathrm{S}}1}}/{P_{{\mathrm{S}}2}}\right) \right e^{-{\mathrm{{\lambda}}}t}\end{equation*}\end{document} and 5\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{equation*}I_{{\mathrm{D}}} \left \left(t\right) \right =1+ \left \left(1-{P_{{\mathrm{D}}1}}/{P_{{\mathrm{D}}2}}\right) \right e^{-2{\mathrm{{\lambda}}}t}- \left \left(2-{P_{{\mathrm{D}}1}}/{P_{{\mathrm{D}}2}}\right) \right e^{-{\mathrm{{\lambda}}}t}{\mathrm{.}}\end{equation*}\end{document} |
View Article: PubMed Central - PubMed
Affiliation: Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.