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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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Slow-gating behaviors of the heterodimer. (A–C) Slow gating of the heterodimer without MTSEA modification. (A) Single-channel trace at −60 mV revealed two current levels and several short inactivation events. (B) Voltage activation experiment to examine slow-gating transitions at the macroscopic current level (n = 3). (C) Temperature-jump experiment revealed prominent inactivation relaxation (n = 3). T1 = 22.5°C, T2 = 28.2°C. Symbols and experimental procedures for B and C were as in Fig. 2E and Fig. F. (D–F) Slow-gating behaviors of the MTSEA-modified heterodimers. (D) Single-channel recording of a MTSEA-modified heterodimer. Membrane potential, −60 mV. (E and F) Voltage and temperature jump experiments (n = 3). Procedures were as in B and C with 30–40 μM MTSEA in the bath. T1 = 21.9°C, T2 = 28.1°C.
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Figure 8: Slow-gating behaviors of the heterodimer. (A–C) Slow gating of the heterodimer without MTSEA modification. (A) Single-channel trace at −60 mV revealed two current levels and several short inactivation events. (B) Voltage activation experiment to examine slow-gating transitions at the macroscopic current level (n = 3). (C) Temperature-jump experiment revealed prominent inactivation relaxation (n = 3). T1 = 22.5°C, T2 = 28.2°C. Symbols and experimental procedures for B and C were as in Fig. 2E and Fig. F. (D–F) Slow-gating behaviors of the MTSEA-modified heterodimers. (D) Single-channel recording of a MTSEA-modified heterodimer. Membrane potential, −60 mV. (E and F) Voltage and temperature jump experiments (n = 3). Procedures were as in B and C with 30–40 μM MTSEA in the bath. T1 = 21.9°C, T2 = 28.1°C.

Mentions: The independence shown above for the fast gating, however, was not observed with respect to inactivation because the latter is influenced by both pores (Ludewig et al. 1996). Without MTSEA, the tandem heterodimer revealed only two current levels with prominent inactivation events (Fig. 8 A), even though the mutation was constructed in the background of C212S. The inactivation process in this one-pore channel was both voltage and temperature dependent (Fig. 8B and Fig. C). With MTSEA modification, the second pore became functional and the open probability of the slow gate of the two-pore channel was greatly increased (Fig. 8, D–F) to a probability similar to that of the K165C* homodimer (Fig. 2E and Fig. F). These results indicate that residue K165 is also important in the slow gating, and the open probability of the slow gate is determined by both pores. The closure of one pore renders the channel easier to inactivate, recapitulating the previously reported nonequilibrium gating cycle of ClC-0 (Richard and Miller 1990).


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)

Slow-gating behaviors of the heterodimer. (A–C) Slow gating of the heterodimer without MTSEA modification. (A) Single-channel trace at −60 mV revealed two current levels and several short inactivation events. (B) Voltage activation experiment to examine slow-gating transitions at the macroscopic current level (n = 3). (C) Temperature-jump experiment revealed prominent inactivation relaxation (n = 3). T1 = 22.5°C, T2 = 28.2°C. Symbols and experimental procedures for B and C were as in Fig. 2E and Fig. F. (D–F) Slow-gating behaviors of the MTSEA-modified heterodimers. (D) Single-channel recording of a MTSEA-modified heterodimer. Membrane potential, −60 mV. (E and F) Voltage and temperature jump experiments (n = 3). Procedures were as in B and C with 30–40 μM MTSEA in the bath. T1 = 21.9°C, T2 = 28.1°C.
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Figure 8: Slow-gating behaviors of the heterodimer. (A–C) Slow gating of the heterodimer without MTSEA modification. (A) Single-channel trace at −60 mV revealed two current levels and several short inactivation events. (B) Voltage activation experiment to examine slow-gating transitions at the macroscopic current level (n = 3). (C) Temperature-jump experiment revealed prominent inactivation relaxation (n = 3). T1 = 22.5°C, T2 = 28.2°C. Symbols and experimental procedures for B and C were as in Fig. 2E and Fig. F. (D–F) Slow-gating behaviors of the MTSEA-modified heterodimers. (D) Single-channel recording of a MTSEA-modified heterodimer. Membrane potential, −60 mV. (E and F) Voltage and temperature jump experiments (n = 3). Procedures were as in B and C with 30–40 μM MTSEA in the bath. T1 = 21.9°C, T2 = 28.1°C.
Mentions: The independence shown above for the fast gating, however, was not observed with respect to inactivation because the latter is influenced by both pores (Ludewig et al. 1996). Without MTSEA, the tandem heterodimer revealed only two current levels with prominent inactivation events (Fig. 8 A), even though the mutation was constructed in the background of C212S. The inactivation process in this one-pore channel was both voltage and temperature dependent (Fig. 8B and Fig. C). With MTSEA modification, the second pore became functional and the open probability of the slow gate of the two-pore channel was greatly increased (Fig. 8, D–F) to a probability similar to that of the K165C* homodimer (Fig. 2E and Fig. F). These results indicate that residue K165 is also important in the slow gating, and the open probability of the slow gate is determined by both pores. The closure of one pore renders the channel easier to inactivate, recapitulating the previously reported nonequilibrium gating cycle of ClC-0 (Richard and Miller 1990).

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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