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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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(A) Single-channel recordings of the MTSPA-modified K165C homodimer at −90 and −110 mV. Dotted lines represent zero-current level. Next to each trace are current amplitude histograms compiled from 30 s (top and bottom) of recording traces containing the 2-s examples on the left. (B) Single-channel recordings of the MTSPA-modified K165C-K165 heterodimer. Amplitude histograms were from 23 s (top) and 30 s (bottom) of recording traces. Capital and small letters represent the pores with big and small conductances, respectively. C, close; O, open. The smaller conductance level in the heterodimer corresponds to the MTSPA-modified pore and is equal to the conductance levels in the homodimer shown in A.
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Figure 4: (A) Single-channel recordings of the MTSPA-modified K165C homodimer at −90 and −110 mV. Dotted lines represent zero-current level. Next to each trace are current amplitude histograms compiled from 30 s (top and bottom) of recording traces containing the 2-s examples on the left. (B) Single-channel recordings of the MTSPA-modified K165C-K165 heterodimer. Amplitude histograms were from 23 s (top) and 30 s (bottom) of recording traces. Capital and small letters represent the pores with big and small conductances, respectively. C, close; O, open. The smaller conductance level in the heterodimer corresponds to the MTSPA-modified pore and is equal to the conductance levels in the homodimer shown in A.

Mentions: As the shift of the Po-V curve was thought to be a pore property, we suspected that K165 may be located in the pore region. To address this possibility more directly, we compared pore properties between K165 and K165C* channels. The blockage by thiocyanate (SCN−), a pore blocker of ClC-0 (White and Miller 1981), was observed in both channels. For the K165 channel, it took 5.5 mM external SCN− to block half of the current at +40 mV, but only 1.3 mM SCN− was needed to reach the same effect in the K165C* channel (Fig. 3A and Fig. B). Examination of the effect of SCN− on single-channel conductance corroborates the apparent blocking affinities derived from macroscopic current recordings in both channels (Fig. 3C and Fig. D), suggesting that K165 is likely to be located near the pore. Another piece of evidence to support the pore location of K165 comes from a reduction in the channel conductance when K165C was modified by MTSPA. In both the K165C homodimer (Fig. 4 A) and K165C-K165 heterodimer (B), the channel conductance of the MTSPA-modified pore was ∼50–60% of that of the wild-type or the MTSEA-modified pore (also see results below). In addition, the MTSPA-modified pores have the same conductance irrespective of the side chain at residue 165 in the other subunit, an observation favoring two independent pores. The MTSPA-modified cysteine has a side chain slightly longer than the MTSEA-modified side chain (Fig. 1 C), a subtle change that influences the rate of Cl− permeation. We have also compared the permeation of various anions in the K165 and K165C* channels. Both channels revealed an anomalous mole fraction effect for mixtures of Cl− and SCN− with a difference in the left arms of the normalized curves (Fig. 5), reflecting different SCN− blocking affinities in these two channels. The anion permeability sequence was SCN− > Cl− > Br− > NO3− > I− for both channels (permeability ratios: K165, 1.20 ± 0.04:1:0.80 ± 0.02:0.66 ± 0.02:0.44 ± 0.04, n = 5; K165C*, 1.22 ± 0.08:1:0.95 ± 0.03:0.74 ± 0.02:0.51 ± 0.05, n = 3–5). The small alteration in ion permeation suggests that the K165C* mutation has not altered too much the global structure of the channel pore.


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)

(A) Single-channel recordings of the MTSPA-modified K165C homodimer at −90 and −110 mV. Dotted lines represent zero-current level. Next to each trace are current amplitude histograms compiled from 30 s (top and bottom) of recording traces containing the 2-s examples on the left. (B) Single-channel recordings of the MTSPA-modified K165C-K165 heterodimer. Amplitude histograms were from 23 s (top) and 30 s (bottom) of recording traces. Capital and small letters represent the pores with big and small conductances, respectively. C, close; O, open. The smaller conductance level in the heterodimer corresponds to the MTSPA-modified pore and is equal to the conductance levels in the homodimer shown in A.
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Related In: Results  -  Collection

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Figure 4: (A) Single-channel recordings of the MTSPA-modified K165C homodimer at −90 and −110 mV. Dotted lines represent zero-current level. Next to each trace are current amplitude histograms compiled from 30 s (top and bottom) of recording traces containing the 2-s examples on the left. (B) Single-channel recordings of the MTSPA-modified K165C-K165 heterodimer. Amplitude histograms were from 23 s (top) and 30 s (bottom) of recording traces. Capital and small letters represent the pores with big and small conductances, respectively. C, close; O, open. The smaller conductance level in the heterodimer corresponds to the MTSPA-modified pore and is equal to the conductance levels in the homodimer shown in A.
Mentions: As the shift of the Po-V curve was thought to be a pore property, we suspected that K165 may be located in the pore region. To address this possibility more directly, we compared pore properties between K165 and K165C* channels. The blockage by thiocyanate (SCN−), a pore blocker of ClC-0 (White and Miller 1981), was observed in both channels. For the K165 channel, it took 5.5 mM external SCN− to block half of the current at +40 mV, but only 1.3 mM SCN− was needed to reach the same effect in the K165C* channel (Fig. 3A and Fig. B). Examination of the effect of SCN− on single-channel conductance corroborates the apparent blocking affinities derived from macroscopic current recordings in both channels (Fig. 3C and Fig. D), suggesting that K165 is likely to be located near the pore. Another piece of evidence to support the pore location of K165 comes from a reduction in the channel conductance when K165C was modified by MTSPA. In both the K165C homodimer (Fig. 4 A) and K165C-K165 heterodimer (B), the channel conductance of the MTSPA-modified pore was ∼50–60% of that of the wild-type or the MTSEA-modified pore (also see results below). In addition, the MTSPA-modified pores have the same conductance irrespective of the side chain at residue 165 in the other subunit, an observation favoring two independent pores. The MTSPA-modified cysteine has a side chain slightly longer than the MTSEA-modified side chain (Fig. 1 C), a subtle change that influences the rate of Cl− permeation. We have also compared the permeation of various anions in the K165 and K165C* channels. Both channels revealed an anomalous mole fraction effect for mixtures of Cl− and SCN− with a difference in the left arms of the normalized curves (Fig. 5), reflecting different SCN− blocking affinities in these two channels. The anion permeability sequence was SCN− > Cl− > Br− > NO3− > I− for both channels (permeability ratios: K165, 1.20 ± 0.04:1:0.80 ± 0.02:0.66 ± 0.02:0.44 ± 0.04, n = 5; K165C*, 1.22 ± 0.08:1:0.95 ± 0.03:0.74 ± 0.02:0.51 ± 0.05, n = 3–5). The small alteration in ion permeation suggests that the K165C* mutation has not altered too much the global structure of the channel pore.

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.

Show MeSH