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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- and slow-gating properties of K165 (filled symbols) and K165C* channels (open symbols) derived from macroscopic current recordings. (A) Comparison of the fast-gate Po-V curves at 100.6 (circles) and 4.6 mM (squares) of [Cl−]o. Solid curves were drawn according to a Boltzmann equation: Po = Pmin + (1 − Pmin)/{1 + exp[−zF(V − V1/2)/RT]}, with z = 0.8–1.2, Pmin = 0.05–0.08. V1/2s in 100.6 and 4.6 mM [Cl−]o were: (K165) −76 and −14 mV; (K165C*) −31 and 35 mV. (B) V1/2s of the fast-gate Po-V curves as a function of [Cl−]o (n = 3–8). (C) Opening rates, α, of the fast gate for K165 and K165C* channels in 100.6 and 4.6 mM [Cl−]o. Symbols are the same as in A (n = 3–8). (D) Opening rates of the channels as a function of [Cl−]o. Data points taken at −40 (•) and −80 mV (▪) for the K165 channel and at 0 (○) and −40 mV (□) for the K165C* channel. Solid and dotted curves were the best fit to the hyperbolic equation, α = αmax[Cl−]o/(K1/2 + [Cl−]o). The fitted αmaxs are (ms−1): K165, 0.29 (−40 mV) and 0.12 (−80 mV); K165C*, 0.12 (0 mV) and 0.04 (−40 mV). The fitted K1/2s are (mM): K165, 107 (−40 mV) and 121 (−80 mV); K165C*, 103 (0 mV) and 108 (−40 mV). (E) Slow-gating transition examined by voltage activation. 30–40 μM MTSEA in the bath solution. Numbers 1 and 2 are the pulsing protocols (see materials and methods) used in the indicated periods (n = 3). Current amplitudes normalized to that of the point right before pulsing protocol 1. Dotted line represents zero-current level. (F) Temperature jump experiment revealed that the probability of closing the slow gate was only minimal upon raising the bath temperature. Pulsing protocol 1. 30–40 μM MTSEA was present. Current amplitudes normalized to that of the first point. T1 = 21.4°C, T2 = 27.5°C (n = 3).
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Figure 2: Fast- and slow-gating properties of K165 (filled symbols) and K165C* channels (open symbols) derived from macroscopic current recordings. (A) Comparison of the fast-gate Po-V curves at 100.6 (circles) and 4.6 mM (squares) of [Cl−]o. Solid curves were drawn according to a Boltzmann equation: Po = Pmin + (1 − Pmin)/{1 + exp[−zF(V − V1/2)/RT]}, with z = 0.8–1.2, Pmin = 0.05–0.08. V1/2s in 100.6 and 4.6 mM [Cl−]o were: (K165) −76 and −14 mV; (K165C*) −31 and 35 mV. (B) V1/2s of the fast-gate Po-V curves as a function of [Cl−]o (n = 3–8). (C) Opening rates, α, of the fast gate for K165 and K165C* channels in 100.6 and 4.6 mM [Cl−]o. Symbols are the same as in A (n = 3–8). (D) Opening rates of the channels as a function of [Cl−]o. Data points taken at −40 (•) and −80 mV (▪) for the K165 channel and at 0 (○) and −40 mV (□) for the K165C* channel. Solid and dotted curves were the best fit to the hyperbolic equation, α = αmax[Cl−]o/(K1/2 + [Cl−]o). The fitted αmaxs are (ms−1): K165, 0.29 (−40 mV) and 0.12 (−80 mV); K165C*, 0.12 (0 mV) and 0.04 (−40 mV). The fitted K1/2s are (mM): K165, 107 (−40 mV) and 121 (−80 mV); K165C*, 103 (0 mV) and 108 (−40 mV). (E) Slow-gating transition examined by voltage activation. 30–40 μM MTSEA in the bath solution. Numbers 1 and 2 are the pulsing protocols (see materials and methods) used in the indicated periods (n = 3). Current amplitudes normalized to that of the point right before pulsing protocol 1. Dotted line represents zero-current level. (F) Temperature jump experiment revealed that the probability of closing the slow gate was only minimal upon raising the bath temperature. Pulsing protocol 1. 30–40 μM MTSEA was present. Current amplitudes normalized to that of the first point. T1 = 21.4°C, T2 = 27.5°C (n = 3).

Mentions: Modification of cysteine by MTSEA converts the side chain to a structure similar to that of a lysine residue (Fig. 1 C), presumably the reason for the reopening of the channel. We thus adopt the name K165C* for the MTSEA-induced functional homodimeric channel with C* representing the MTSEA-modified cysteine. The fast gating of K165C* was similar to that of the wild-type channel containing lysine at the 165 position, with current deactivation at −150 mV (Fig. 1 A). However, the Po-V curve of K165C* was right shifted by 45 mV when compared with that of the K165 channel at an external Cl− concentration ([Cl−]o) of ∼100 mM (Fig. 2 A). This curve was further shifted towards a more depolarized membrane potential in response to a low [Cl−]o. The V1/2 of the Po-V curves in both channels had a more negative value at higher [Cl−]o, but the effect was saturated at [Cl−]o > 150–300 mM. Furthermore, there was always an ∼40–50-mV difference in V1/2 between two channels at the same [Cl−]o (Fig. 2 B). This difference was mostly due to a shift of the opening rate curve of K165C* towards a more depolarized membrane potential (Fig. 2 C), an effect similar to that of lowering [Cl−]o on the wild-type channel (Pusch et al. 1995; Chen and Miller 1996). Since the ability of external Cl− to shift the fast-gate Po-V curve is mediated by the Cl−-binding sites in the pore, we analyzed the [Cl−]o dependence of the fast-gate opening rate derived from macroscopic current recordings (Fig. 2 D). Curve fitting to hyperbolic equations suggested that the apparent affinity for Cl− binding (Chen and Miller 1996) was not changed. Rather, the effect may result from a higher energy barrier in the subsequent Cl− translocation process that drives the bound Cl− to open the channel (Chen and Miller 1996). In addition to the fast-gating properties, we also examined the slow gating of K165C*. Both the voltage (Fig. 2 E) and temperature (F) changes resulted in almost no change in the open probability of the slow gate, a phenomenon most likely resulting from the C212S mutation (Lin et al. 1999). As will be shown later, the little inactivation in this channel provides a well defined slow-gating behavior that is important in interpreting the apparent MTS modification rate.


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- and slow-gating properties of K165 (filled symbols) and K165C* channels (open symbols) derived from macroscopic current recordings. (A) Comparison of the fast-gate Po-V curves at 100.6 (circles) and 4.6 mM (squares) of [Cl−]o. Solid curves were drawn according to a Boltzmann equation: Po = Pmin + (1 − Pmin)/{1 + exp[−zF(V − V1/2)/RT]}, with z = 0.8–1.2, Pmin = 0.05–0.08. V1/2s in 100.6 and 4.6 mM [Cl−]o were: (K165) −76 and −14 mV; (K165C*) −31 and 35 mV. (B) V1/2s of the fast-gate Po-V curves as a function of [Cl−]o (n = 3–8). (C) Opening rates, α, of the fast gate for K165 and K165C* channels in 100.6 and 4.6 mM [Cl−]o. Symbols are the same as in A (n = 3–8). (D) Opening rates of the channels as a function of [Cl−]o. Data points taken at −40 (•) and −80 mV (▪) for the K165 channel and at 0 (○) and −40 mV (□) for the K165C* channel. Solid and dotted curves were the best fit to the hyperbolic equation, α = αmax[Cl−]o/(K1/2 + [Cl−]o). The fitted αmaxs are (ms−1): K165, 0.29 (−40 mV) and 0.12 (−80 mV); K165C*, 0.12 (0 mV) and 0.04 (−40 mV). The fitted K1/2s are (mM): K165, 107 (−40 mV) and 121 (−80 mV); K165C*, 103 (0 mV) and 108 (−40 mV). (E) Slow-gating transition examined by voltage activation. 30–40 μM MTSEA in the bath solution. Numbers 1 and 2 are the pulsing protocols (see materials and methods) used in the indicated periods (n = 3). Current amplitudes normalized to that of the point right before pulsing protocol 1. Dotted line represents zero-current level. (F) Temperature jump experiment revealed that the probability of closing the slow gate was only minimal upon raising the bath temperature. Pulsing protocol 1. 30–40 μM MTSEA was present. Current amplitudes normalized to that of the first point. T1 = 21.4°C, T2 = 27.5°C (n = 3).
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Related In: Results  -  Collection

Show All Figures
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Figure 2: Fast- and slow-gating properties of K165 (filled symbols) and K165C* channels (open symbols) derived from macroscopic current recordings. (A) Comparison of the fast-gate Po-V curves at 100.6 (circles) and 4.6 mM (squares) of [Cl−]o. Solid curves were drawn according to a Boltzmann equation: Po = Pmin + (1 − Pmin)/{1 + exp[−zF(V − V1/2)/RT]}, with z = 0.8–1.2, Pmin = 0.05–0.08. V1/2s in 100.6 and 4.6 mM [Cl−]o were: (K165) −76 and −14 mV; (K165C*) −31 and 35 mV. (B) V1/2s of the fast-gate Po-V curves as a function of [Cl−]o (n = 3–8). (C) Opening rates, α, of the fast gate for K165 and K165C* channels in 100.6 and 4.6 mM [Cl−]o. Symbols are the same as in A (n = 3–8). (D) Opening rates of the channels as a function of [Cl−]o. Data points taken at −40 (•) and −80 mV (▪) for the K165 channel and at 0 (○) and −40 mV (□) for the K165C* channel. Solid and dotted curves were the best fit to the hyperbolic equation, α = αmax[Cl−]o/(K1/2 + [Cl−]o). The fitted αmaxs are (ms−1): K165, 0.29 (−40 mV) and 0.12 (−80 mV); K165C*, 0.12 (0 mV) and 0.04 (−40 mV). The fitted K1/2s are (mM): K165, 107 (−40 mV) and 121 (−80 mV); K165C*, 103 (0 mV) and 108 (−40 mV). (E) Slow-gating transition examined by voltage activation. 30–40 μM MTSEA in the bath solution. Numbers 1 and 2 are the pulsing protocols (see materials and methods) used in the indicated periods (n = 3). Current amplitudes normalized to that of the point right before pulsing protocol 1. Dotted line represents zero-current level. (F) Temperature jump experiment revealed that the probability of closing the slow gate was only minimal upon raising the bath temperature. Pulsing protocol 1. 30–40 μM MTSEA was present. Current amplitudes normalized to that of the first point. T1 = 21.4°C, T2 = 27.5°C (n = 3).
Mentions: Modification of cysteine by MTSEA converts the side chain to a structure similar to that of a lysine residue (Fig. 1 C), presumably the reason for the reopening of the channel. We thus adopt the name K165C* for the MTSEA-induced functional homodimeric channel with C* representing the MTSEA-modified cysteine. The fast gating of K165C* was similar to that of the wild-type channel containing lysine at the 165 position, with current deactivation at −150 mV (Fig. 1 A). However, the Po-V curve of K165C* was right shifted by 45 mV when compared with that of the K165 channel at an external Cl− concentration ([Cl−]o) of ∼100 mM (Fig. 2 A). This curve was further shifted towards a more depolarized membrane potential in response to a low [Cl−]o. The V1/2 of the Po-V curves in both channels had a more negative value at higher [Cl−]o, but the effect was saturated at [Cl−]o > 150–300 mM. Furthermore, there was always an ∼40–50-mV difference in V1/2 between two channels at the same [Cl−]o (Fig. 2 B). This difference was mostly due to a shift of the opening rate curve of K165C* towards a more depolarized membrane potential (Fig. 2 C), an effect similar to that of lowering [Cl−]o on the wild-type channel (Pusch et al. 1995; Chen and Miller 1996). Since the ability of external Cl− to shift the fast-gate Po-V curve is mediated by the Cl−-binding sites in the pore, we analyzed the [Cl−]o dependence of the fast-gate opening rate derived from macroscopic current recordings (Fig. 2 D). Curve fitting to hyperbolic equations suggested that the apparent affinity for Cl− binding (Chen and Miller 1996) was not changed. Rather, the effect may result from a higher energy barrier in the subsequent Cl− translocation process that drives the bound Cl− to open the channel (Chen and Miller 1996). In addition to the fast-gating properties, we also examined the slow gating of K165C*. Both the voltage (Fig. 2 E) and temperature (F) changes resulted in almost no change in the open probability of the slow gate, a phenomenon most likely resulting from the C212S mutation (Lin et al. 1999). As will be shown later, the little inactivation in this channel provides a well defined slow-gating behavior that is important in interpreting the apparent MTS modification rate.

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