Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparison of the SCN− block between K165 and K165C* channels. (A) SCN− block of the whole oocyte current. Pulsing protocol 2. Numbers indicate the SCN− concentrations (millimolar) in the bath solution. Dotted lines are zero-current level. Vertical scale bar represents 12 and 2 μA for K165 and K165C*, respectively. (B) The K165C* channel was more sensitive to the SCN− block. Data points derived from experiments like those in A. Solid curves were drawn according to: Inorm = I∞ + (1 − I∞)/(1 + [SCN−]/K1/2), with values of K1/2 and I∞: (K165) 5.5 mM and 0.21; (K165C*) 1.3 mM and 0.27 (n = 4). (C) Single-channel recording of the SCN−-blocked K165 and K165C* channels at +40 mV. Symmetrical solutions on both sides of the membrane except that the indicated concentrations of NaSCN were added in the pipette solution. Dotted lines are zero-current level. (D) Averaged single-pore conductance at +40 mV (n = 3–8). The two current levels in traces like those shown in C were determined from all-points amplitude histograms and the difference in current was divided by two to calculate the conductance of one pore. Solid curves are the same curves from B after multiplying the respective channel conductance in the absence of SCN−.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230621&req=5

Figure 3: Comparison of the SCN− block between K165 and K165C* channels. (A) SCN− block of the whole oocyte current. Pulsing protocol 2. Numbers indicate the SCN− concentrations (millimolar) in the bath solution. Dotted lines are zero-current level. Vertical scale bar represents 12 and 2 μA for K165 and K165C*, respectively. (B) The K165C* channel was more sensitive to the SCN− block. Data points derived from experiments like those in A. Solid curves were drawn according to: Inorm = I∞ + (1 − I∞)/(1 + [SCN−]/K1/2), with values of K1/2 and I∞: (K165) 5.5 mM and 0.21; (K165C*) 1.3 mM and 0.27 (n = 4). (C) Single-channel recording of the SCN−-blocked K165 and K165C* channels at +40 mV. Symmetrical solutions on both sides of the membrane except that the indicated concentrations of NaSCN were added in the pipette solution. Dotted lines are zero-current level. (D) Averaged single-pore conductance at +40 mV (n = 3–8). The two current levels in traces like those shown in C were determined from all-points amplitude histograms and the difference in current was divided by two to calculate the conductance of one pore. Solid curves are the same curves from B after multiplying the respective channel conductance in the absence of SCN−.

Mentions: Procedures for obtaining inside-out patches were as previously described (Lin et al. 1999). Except where indicated, the pipette (extracellular) solution was (mM): 110 NMDG-Cl, 5 MgCl2, 1 CaCl2, 5 HEPES, pH 7.6. The bath (intracellular) solution contained (mM): 110 NaCl, 5 MgCl2, 1 EGTA, 5 HEPES, pH 7.6. The current, filtered at 200 Hz (−3 dB corner frequency, four-pole Bessel), was digitized by an acquisition board (DAP 800; Microstar) at 1 kHz with home-made software (Chen and Miller 1996; Lin et al. 1999). In the SCN− blocking experiments (see Fig. 3C and Fig. D), both sides of the membrane patch have the same solution containing (mM): 110 NMDG-Cl, 5 MgCl2, 1 EGTA, 5 HEPES, pH 7.6, with the indicated concentrations of NaSCN being added to the pipette solution. The presence of Na+ or NMDG+ on either side of the membrane did not significantly affect the single-channel conductance or the gating properties.


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)

Comparison of the SCN− block between K165 and K165C* channels. (A) SCN− block of the whole oocyte current. Pulsing protocol 2. Numbers indicate the SCN− concentrations (millimolar) in the bath solution. Dotted lines are zero-current level. Vertical scale bar represents 12 and 2 μA for K165 and K165C*, respectively. (B) The K165C* channel was more sensitive to the SCN− block. Data points derived from experiments like those in A. Solid curves were drawn according to: Inorm = I∞ + (1 − I∞)/(1 + [SCN−]/K1/2), with values of K1/2 and I∞: (K165) 5.5 mM and 0.21; (K165C*) 1.3 mM and 0.27 (n = 4). (C) Single-channel recording of the SCN−-blocked K165 and K165C* channels at +40 mV. Symmetrical solutions on both sides of the membrane except that the indicated concentrations of NaSCN were added in the pipette solution. Dotted lines are zero-current level. (D) Averaged single-pore conductance at +40 mV (n = 3–8). The two current levels in traces like those shown in C were determined from all-points amplitude histograms and the difference in current was divided by two to calculate the conductance of one pore. Solid curves are the same curves from B after multiplying the respective channel conductance in the absence of SCN−.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230621&req=5

Figure 3: Comparison of the SCN− block between K165 and K165C* channels. (A) SCN− block of the whole oocyte current. Pulsing protocol 2. Numbers indicate the SCN− concentrations (millimolar) in the bath solution. Dotted lines are zero-current level. Vertical scale bar represents 12 and 2 μA for K165 and K165C*, respectively. (B) The K165C* channel was more sensitive to the SCN− block. Data points derived from experiments like those in A. Solid curves were drawn according to: Inorm = I∞ + (1 − I∞)/(1 + [SCN−]/K1/2), with values of K1/2 and I∞: (K165) 5.5 mM and 0.21; (K165C*) 1.3 mM and 0.27 (n = 4). (C) Single-channel recording of the SCN−-blocked K165 and K165C* channels at +40 mV. Symmetrical solutions on both sides of the membrane except that the indicated concentrations of NaSCN were added in the pipette solution. Dotted lines are zero-current level. (D) Averaged single-pore conductance at +40 mV (n = 3–8). The two current levels in traces like those shown in C were determined from all-points amplitude histograms and the difference in current was divided by two to calculate the conductance of one pore. Solid curves are the same curves from B after multiplying the respective channel conductance in the absence of SCN−.
Mentions: Procedures for obtaining inside-out patches were as previously described (Lin et al. 1999). Except where indicated, the pipette (extracellular) solution was (mM): 110 NMDG-Cl, 5 MgCl2, 1 CaCl2, 5 HEPES, pH 7.6. The bath (intracellular) solution contained (mM): 110 NaCl, 5 MgCl2, 1 EGTA, 5 HEPES, pH 7.6. The current, filtered at 200 Hz (−3 dB corner frequency, four-pole Bessel), was digitized by an acquisition board (DAP 800; Microstar) at 1 kHz with home-made software (Chen and Miller 1996; Lin et al. 1999). In the SCN− blocking experiments (see Fig. 3C and Fig. D), both sides of the membrane patch have the same solution containing (mM): 110 NMDG-Cl, 5 MgCl2, 1 EGTA, 5 HEPES, pH 7.6, with the indicated concentrations of NaSCN being added to the pipette solution. The presence of Na+ or NMDG+ on either side of the membrane did not significantly affect the single-channel conductance or the gating properties.

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