Lin CW and Chen TY (2000), Cysteine modification of a putative pore residu...

XB-ART-10273

J Gen Physiol 2000 Oct 01;1164:535-46.

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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.

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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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Species referenced: Xenopus
Genes referenced: clcn1 spata2 tbxt.2

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	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−.
	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.
	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).
	Figure 4. (A) Single-channel recordings of the MTSPA-modified K165C homodimer at −90 and −110 mV. Dotted lines represent zero-current level. Next to each trace are current amplitude histograms compiled from 30 s (top and bottom) of recording traces containing the 2-s examples on the left. (B) Single-channel recordings of the MTSPA-modified K165C-K165 heterodimer. Amplitude histograms were from 23 s (top) and 30 s (bottom) of recording traces. Capital and small letters represent the pores with big and small conductances, respectively. C, close; O, open. The smaller conductance level in the heterodimer corresponds to the MTSPA-modified pore and is equal to the conductance levels in the homodimer shown in A.
	Figure 5. Comparison of the anomalous mole fraction effect for mixtures of Cl− and SCN−. All measured currents were normalized to the current obtained in the absence of NaSCN. Each point represents the average of four measurements. *Significantly different (P < 0.05, Student's t test) from the K165 channel.
	Figure 6. Fast-gating properties of heterodimers after MTSEA modification. (A) Single-channel recording traces of K165C, K165-K165C, and K165 channels. Membrane potential, −40 mV. Dotted lines are zero-current level. (B) Po-V curves derived from single-channel recordings (n = 3–7). Solid curves are Boltzmann curves for K165 (○) and K165C* (•) channels, while the dashed curve is simply the average of the two solid curves. (□) The measured Po of the heterodimers (average of K165-K165C* and K165C-K165). (C) Single-channel recordings of the heterodimer K165C-K165 at three different voltages. Open probabilities shown on the right were calculated from >40-s continuous recordings, including the three traces shown at left. (D) Comparison of the measured state probabilities (bars) with the expected state probabilities (circles). (•) Calculated from the averaged Po of the heterodimer according to (binomial distribution). (○) Calculated according to , assuming distinct Po's for two pores (multinomial distribution). The open probabilities were taken from the Po's of the homodimers at the corresponding voltages shown in B. Statistical analysis showed significant difference (P < 0.01, one sample t test, SPSS 8.0; SPSS, Inc.) between the measured probabilities of the M level and the expected values derived from the binomial model in all three voltages. The comparisons of the measured probabilities with the expected values from the multinomial model, however, showed no difference (P > 0.01).
	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.
	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.
	Figure 9. Independence of MTSEA modification on the introduced cysteines. (A) Reaction schemes for MTSEA modification. When the modification rates are smaller than the slow-gating transition rates, the schemes can be reduced to linear models. (B) Slow-gate open probability contributes to the apparent MTSEA-modification rate. (Left) Calculated current-induction curves of the homodimer () when PD1/PD2 are 0, 0.5, and 1, respectively. The normalized curve of the heterodimer () is the same as that of the homodimer when PD1/PD2 = 1. (Right) Ratios of the time constants derived from single-exponential curve fittings plotted against PD1/PD2. (C) Experimental comparison of the apparent modification rates between hetero- and homodimeric channels. (Left) Current induction in the heterodimer (□, n = 6) and homodimer (○, n = 3) by 3 μM MTSEA. All current amplitudes, after subtraction by the current before applying MTSEA, were normalized to the maximal current induced by MTSEA. One error bar plotted in every 10 points. Time constants were 49.3 and 80.7 s for the hetero- and homodimer, respectively. (Right) Current-induction rates in the homodimeric (○) and heterodimeric channels (□) at various concentrations of MTSEA. The slopes and y intercepts of the solid lines were: (heterodimer) 7.2 × 103 M−1s−1 and 0.0037 s−1; (homodimer) 4.6 × 103 M−1s−1 and 0.0032 s−1. (Inset) Ratios of the induction time constants of the homodimer to those of the heterodimer.

References [+] :

Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed, Xenbase