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Cysteine modification of a putative pore residue in ClC-0: implication for the pore stoichiometry of ClC chloride channels.

Lin CW, Chen TY - J. Gen. Physiol. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.

ABSTRACT
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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Fast-gating properties of heterodimers after MTSEA modification. (A) Single-channel recording traces of K165C*, K165-K165C*, and K165 channels. Membrane potential, −40 mV. Dotted lines are zero-current level. (B) Po-V curves derived from single-channel recordings (n = 3–7). Solid curves are Boltzmann curves for K165 (○) and K165C* (•) channels, while the dashed curve is simply the average of the two solid curves. (□) The measured Po of the heterodimers (average of K165-K165C* and K165C*-K165). (C) Single-channel recordings of the heterodimer K165C*-K165 at three different voltages. Open probabilities shown on the right were calculated from >40-s continuous recordings, including the three traces shown at left. (D) Comparison of the measured state probabilities (bars) with the expected state probabilities (circles). (•) Calculated from the averaged Po of the heterodimer according to  (binomial distribution). (○) Calculated according to , assuming distinct Po's for two pores (multinomial distribution). The open probabilities were taken from the Po's of the homodimers at the corresponding voltages shown in B. Statistical analysis showed significant difference (P < 0.01, one sample t test, SPSS 8.0; SPSS, Inc.) between the measured probabilities of the M level and the expected values derived from the binomial model in all three voltages. The comparisons of the measured probabilities with the expected values from the multinomial model, however, showed no difference (P > 0.01).
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Figure 6: Fast-gating properties of heterodimers after MTSEA modification. (A) Single-channel recording traces of K165C*, K165-K165C*, and K165 channels. Membrane potential, −40 mV. Dotted lines are zero-current level. (B) Po-V curves derived from single-channel recordings (n = 3–7). Solid curves are Boltzmann curves for K165 (○) and K165C* (•) channels, while the dashed curve is simply the average of the two solid curves. (□) The measured Po of the heterodimers (average of K165-K165C* and K165C*-K165). (C) Single-channel recordings of the heterodimer K165C*-K165 at three different voltages. Open probabilities shown on the right were calculated from >40-s continuous recordings, including the three traces shown at left. (D) Comparison of the measured state probabilities (bars) with the expected state probabilities (circles). (•) Calculated from the averaged Po of the heterodimer according to (binomial distribution). (○) Calculated according to , assuming distinct Po's for two pores (multinomial distribution). The open probabilities were taken from the Po's of the homodimers at the corresponding voltages shown in B. Statistical analysis showed significant difference (P < 0.01, one sample t test, SPSS 8.0; SPSS, Inc.) between the measured probabilities of the M level and the expected values derived from the binomial model in all three voltages. The comparisons of the measured probabilities with the expected values from the multinomial model, however, showed no difference (P > 0.01).

Mentions: To analyze a two-pore channel (see Fig. 6A and Fig. B), the absolute open probability of the channel (average of two pores) was determined from the probabilities of three equally spaced conductance levels, D (down), M (middle), and U (upper) (): 1\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*}P_{{\mathrm{o}}}={f_{{\mathrm{M}}}}/{2}+f_{{\mathrm{U}}}{\mathrm{,}}\end{equation*}\end{document} where fM and fU were the measured probabilities at the M and U levels, respectively. When only two levels were present, as in the one-pore channel, Po was determined, after inactivation events were removed, as the probability of the open level.


Cysteine modification of a putative pore residue in ClC-0: implication for the pore stoichiometry of ClC chloride channels.

Lin CW, Chen TY - J. Gen. Physiol. (2000)

Fast-gating properties of heterodimers after MTSEA modification. (A) Single-channel recording traces of K165C*, K165-K165C*, and K165 channels. Membrane potential, −40 mV. Dotted lines are zero-current level. (B) Po-V curves derived from single-channel recordings (n = 3–7). Solid curves are Boltzmann curves for K165 (○) and K165C* (•) channels, while the dashed curve is simply the average of the two solid curves. (□) The measured Po of the heterodimers (average of K165-K165C* and K165C*-K165). (C) Single-channel recordings of the heterodimer K165C*-K165 at three different voltages. Open probabilities shown on the right were calculated from >40-s continuous recordings, including the three traces shown at left. (D) Comparison of the measured state probabilities (bars) with the expected state probabilities (circles). (•) Calculated from the averaged Po of the heterodimer according to  (binomial distribution). (○) Calculated according to , assuming distinct Po's for two pores (multinomial distribution). The open probabilities were taken from the Po's of the homodimers at the corresponding voltages shown in B. Statistical analysis showed significant difference (P < 0.01, one sample t test, SPSS 8.0; SPSS, Inc.) between the measured probabilities of the M level and the expected values derived from the binomial model in all three voltages. The comparisons of the measured probabilities with the expected values from the multinomial model, however, showed no difference (P > 0.01).
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Figure 6: Fast-gating properties of heterodimers after MTSEA modification. (A) Single-channel recording traces of K165C*, K165-K165C*, and K165 channels. Membrane potential, −40 mV. Dotted lines are zero-current level. (B) Po-V curves derived from single-channel recordings (n = 3–7). Solid curves are Boltzmann curves for K165 (○) and K165C* (•) channels, while the dashed curve is simply the average of the two solid curves. (□) The measured Po of the heterodimers (average of K165-K165C* and K165C*-K165). (C) Single-channel recordings of the heterodimer K165C*-K165 at three different voltages. Open probabilities shown on the right were calculated from >40-s continuous recordings, including the three traces shown at left. (D) Comparison of the measured state probabilities (bars) with the expected state probabilities (circles). (•) Calculated from the averaged Po of the heterodimer according to (binomial distribution). (○) Calculated according to , assuming distinct Po's for two pores (multinomial distribution). The open probabilities were taken from the Po's of the homodimers at the corresponding voltages shown in B. Statistical analysis showed significant difference (P < 0.01, one sample t test, SPSS 8.0; SPSS, Inc.) between the measured probabilities of the M level and the expected values derived from the binomial model in all three voltages. The comparisons of the measured probabilities with the expected values from the multinomial model, however, showed no difference (P > 0.01).
Mentions: To analyze a two-pore channel (see Fig. 6A and Fig. B), the absolute open probability of the channel (average of two pores) was determined from the probabilities of three equally spaced conductance levels, D (down), M (middle), and U (upper) (): 1\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*}P_{{\mathrm{o}}}={f_{{\mathrm{M}}}}/{2}+f_{{\mathrm{U}}}{\mathrm{,}}\end{equation*}\end{document} where fM and fU were the measured probabilities at the M and U levels, respectively. When only two levels were present, as in the one-pore channel, Po was determined, after inactivation events were removed, as the probability of the open level.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.

ABSTRACT
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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