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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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Modification by MTS reagents of K165C/C212S channels expressed in Xenopus oocytes. (A) Functional current induced by MTSEA. Repeated pulsing protocol 2, given one pulse every 2 s. Reagents applied as indicated by horizontal lines. (○) Current amplitudes measured at +40 mV. Examples of the current traces before and after MTSEA, and finally after dithiothreitol treatment are shown. Dotted lines are zero-current level. (B) Reduction of MTSEA-induced current after washout of the modifying reagent. Repeated pulsing protocol 1. Temperature, 20°C. (C) Side-chain structure of lysine and those of cysteine modified with various MTS reagents. (D) Modification of K165C by MTSET without generating functional channels. Pulsing protocol 1 used in this experiment. MTSES produced similar effects in blocking the current induction by MTSEA (data not shown). (E) Current induction in K165C channel by MTSPA. Dashed and solid curves represent the average of three recording traces before (control) and after the application of 0.2 mM MTSPA. Dotted line is zero-current level.
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Figure 1: Modification by MTS reagents of K165C/C212S channels expressed in Xenopus oocytes. (A) Functional current induced by MTSEA. Repeated pulsing protocol 2, given one pulse every 2 s. Reagents applied as indicated by horizontal lines. (○) Current amplitudes measured at +40 mV. Examples of the current traces before and after MTSEA, and finally after dithiothreitol treatment are shown. Dotted lines are zero-current level. (B) Reduction of MTSEA-induced current after washout of the modifying reagent. Repeated pulsing protocol 1. Temperature, 20°C. (C) Side-chain structure of lysine and those of cysteine modified with various MTS reagents. (D) Modification of K165C by MTSET without generating functional channels. Pulsing protocol 1 used in this experiment. MTSES produced similar effects in blocking the current induction by MTSEA (data not shown). (E) Current induction in K165C channel by MTSPA. Dashed and solid curves represent the average of three recording traces before (control) and after the application of 0.2 mM MTSPA. Dotted line is zero-current level.

Mentions: An example of current induction of K165C by MTSEA is shown in Fig. 1 A. Upon the application of MTSEA in the bath solution, ClC-0-like current can be induced. This MTSEA-induced current can be reversed slowly by washing out the modifying reagent, with a current reduction time constant of ∼30 min at ∼20°C (Fig. 1 B). A reducing reagent, dithiothreitol, speeded up this reversing process (Fig. 1 A), indicating that the effect was through a sulfhydryl group. The modification was specific to the introduced cysteine because MTSEA had no effect on wild-type ClC-0 or C212S (data not shown). Another two MTS reagents, 2-(trimethylammonium)ethyl MTS (MTSET) and 2-sulphonatoethyl MTS (MTSES) (Akabas et al. 1992; Stauffer and Karlin 1994), did not induce current, but both compounds blocked the effect of MTSEA (Fig. 1 D). Therefore, these two reagents can also modify the introduced cysteine without generating functional channels. We have also used 3-aminopropyl MTS (MTSPA) to modify the channel. This attaches a slightly longer side chain to cysteine than that of MTSEA modification (see Fig. 1 C for the side-chain structures after modifications by MTS reagents), and MTSPA can also induce current in the K165C mutant (Fig. 1 E).


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)

Modification by MTS reagents of K165C/C212S channels expressed in Xenopus oocytes. (A) Functional current induced by MTSEA. Repeated pulsing protocol 2, given one pulse every 2 s. Reagents applied as indicated by horizontal lines. (○) Current amplitudes measured at +40 mV. Examples of the current traces before and after MTSEA, and finally after dithiothreitol treatment are shown. Dotted lines are zero-current level. (B) Reduction of MTSEA-induced current after washout of the modifying reagent. Repeated pulsing protocol 1. Temperature, 20°C. (C) Side-chain structure of lysine and those of cysteine modified with various MTS reagents. (D) Modification of K165C by MTSET without generating functional channels. Pulsing protocol 1 used in this experiment. MTSES produced similar effects in blocking the current induction by MTSEA (data not shown). (E) Current induction in K165C channel by MTSPA. Dashed and solid curves represent the average of three recording traces before (control) and after the application of 0.2 mM MTSPA. Dotted line is zero-current level.
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Related In: Results  -  Collection

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Figure 1: Modification by MTS reagents of K165C/C212S channels expressed in Xenopus oocytes. (A) Functional current induced by MTSEA. Repeated pulsing protocol 2, given one pulse every 2 s. Reagents applied as indicated by horizontal lines. (○) Current amplitudes measured at +40 mV. Examples of the current traces before and after MTSEA, and finally after dithiothreitol treatment are shown. Dotted lines are zero-current level. (B) Reduction of MTSEA-induced current after washout of the modifying reagent. Repeated pulsing protocol 1. Temperature, 20°C. (C) Side-chain structure of lysine and those of cysteine modified with various MTS reagents. (D) Modification of K165C by MTSET without generating functional channels. Pulsing protocol 1 used in this experiment. MTSES produced similar effects in blocking the current induction by MTSEA (data not shown). (E) Current induction in K165C channel by MTSPA. Dashed and solid curves represent the average of three recording traces before (control) and after the application of 0.2 mM MTSPA. Dotted line is zero-current level.
Mentions: An example of current induction of K165C by MTSEA is shown in Fig. 1 A. Upon the application of MTSEA in the bath solution, ClC-0-like current can be induced. This MTSEA-induced current can be reversed slowly by washing out the modifying reagent, with a current reduction time constant of ∼30 min at ∼20°C (Fig. 1 B). A reducing reagent, dithiothreitol, speeded up this reversing process (Fig. 1 A), indicating that the effect was through a sulfhydryl group. The modification was specific to the introduced cysteine because MTSEA had no effect on wild-type ClC-0 or C212S (data not shown). Another two MTS reagents, 2-(trimethylammonium)ethyl MTS (MTSET) and 2-sulphonatoethyl MTS (MTSES) (Akabas et al. 1992; Stauffer and Karlin 1994), did not induce current, but both compounds blocked the effect of MTSEA (Fig. 1 D). Therefore, these two reagents can also modify the introduced cysteine without generating functional channels. We have also used 3-aminopropyl MTS (MTSPA) to modify the channel. This attaches a slightly longer side chain to cysteine than that of MTSEA modification (see Fig. 1 C for the side-chain structures after modifications by MTS reagents), and MTSPA can also induce current in the K165C mutant (Fig. 1 E).

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
Related in: MedlinePlus