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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 of the heterodimer without MTSEA modification. (A) Single-channel recording traces of the heterodimer K165C-K165 at different voltages. Dotted lines represent zero-current level. (B) Steady state Po-V curve of the single-pore heterodimer. (○) The average of three to eight measurements from traces like those shown in A. Solid and dotted curves are the same Boltzmann curves for the K165 and K165C* homodimers, respectively, as those shown in Fig. 6 B.
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Figure 7: Fast gating of the heterodimer without MTSEA modification. (A) Single-channel recording traces of the heterodimer K165C-K165 at different voltages. Dotted lines represent zero-current level. (B) Steady state Po-V curve of the single-pore heterodimer. (○) The average of three to eight measurements from traces like those shown in A. Solid and dotted curves are the same Boltzmann curves for the K165 and K165C* homodimers, respectively, as those shown in Fig. 6 B.

Mentions: With tandem heterodimers containing K165 in one subunit and K165C in the other, the functional role of K165 in ClC-0 gating can be further characterized. The MTSEA-modified tandem heterodimers revealed a double-barrel–like structure, as with K165 or K165C* homodimers (Fig. 6 A). The average Po of the two pores in either configurations (K165C*-K165 or K165-K165C*) was close to the mean of those of the K165 and K165C* homodimers (Fig. 6 B). However, the Po of each individual pore was different from that of the other pore. For example, the probabilities of the three current levels showed a multinomial distribution when the Po of one pore was equal to the Po of K165 and the other to that of K165C* channels (Fig. 6C and Fig. D). Furthermore, when recording single MTSEA-modified heterodimeric channels, we frequently observed a transition from three to two current levels, presumably due to the loss of the modifying group. Subsequent examination of such a two-level trace always revealed a Po close to that of the K165 channel (Fig. 7). These results together indicate that the two pores of the MTSEA-modified heterodimer have equal conductance and different Po; nevertheless, the principle of independent gating is still preserved.


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 of the heterodimer without MTSEA modification. (A) Single-channel recording traces of the heterodimer K165C-K165 at different voltages. Dotted lines represent zero-current level. (B) Steady state Po-V curve of the single-pore heterodimer. (○) The average of three to eight measurements from traces like those shown in A. Solid and dotted curves are the same Boltzmann curves for the K165 and K165C* homodimers, respectively, as those shown in Fig. 6 B.
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Figure 7: Fast gating of the heterodimer without MTSEA modification. (A) Single-channel recording traces of the heterodimer K165C-K165 at different voltages. Dotted lines represent zero-current level. (B) Steady state Po-V curve of the single-pore heterodimer. (○) The average of three to eight measurements from traces like those shown in A. Solid and dotted curves are the same Boltzmann curves for the K165 and K165C* homodimers, respectively, as those shown in Fig. 6 B.
Mentions: With tandem heterodimers containing K165 in one subunit and K165C in the other, the functional role of K165 in ClC-0 gating can be further characterized. The MTSEA-modified tandem heterodimers revealed a double-barrel–like structure, as with K165 or K165C* homodimers (Fig. 6 A). The average Po of the two pores in either configurations (K165C*-K165 or K165-K165C*) was close to the mean of those of the K165 and K165C* homodimers (Fig. 6 B). However, the Po of each individual pore was different from that of the other pore. For example, the probabilities of the three current levels showed a multinomial distribution when the Po of one pore was equal to the Po of K165 and the other to that of K165C* channels (Fig. 6C and Fig. D). Furthermore, when recording single MTSEA-modified heterodimeric channels, we frequently observed a transition from three to two current levels, presumably due to the loss of the modifying group. Subsequent examination of such a two-level trace always revealed a Po close to that of the K165 channel (Fig. 7). These results together indicate that the two pores of the MTSEA-modified heterodimer have equal conductance and different Po; nevertheless, the principle of independent gating is still preserved.

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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