
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 zerocurrent 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) Singlechannel 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 zerocurrent level. (D) Averaged singlepore conductance at +40 mV (n = 3–8). The two current levels in traces like those shown in C were determined from allpoints 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 zerocurrent level. (B) Reduction of MTSEAinduced current after washout of the modifying reagent. Repeated pulsing protocol 1. Temperature, 20°C. (C) Sidechain 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 zerocurrent level.


Figure 2. Fast and slowgating properties of K165 (filled symbols) and K165C* channels (open symbols) derived from macroscopic current recordings. (A) Comparison of the fastgate PoV 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 fastgate PoV 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) Slowgating 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 zerocurrent 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) Singlechannel recordings of the MTSPAmodified K165C homodimer at −90 and −110 mV. Dotted lines represent zerocurrent level. Next to each trace are current amplitude histograms compiled from 30 s (top and bottom) of recording traces containing the 2s examples on the left. (B) Singlechannel recordings of the MTSPAmodified K165CK165 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 MTSPAmodified 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. Fastgating properties of heterodimers after MTSEA modification. (A) Singlechannel recording traces of K165C*, K165K165C*, and K165 channels. Membrane potential, −40 mV. Dotted lines are zerocurrent level. (B) PoV curves derived from singlechannel 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 K165K165C* and K165C*K165). (C) Singlechannel recordings of the heterodimer K165C*K165 at three different voltages. Open probabilities shown on the right were calculated from >40s 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) Singlechannel recording traces of the heterodimer K165CK165 at different voltages. Dotted lines represent zerocurrent level. (B) Steady state PoV curve of the singlepore 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. Slowgating behaviors of the heterodimer. (A–C) Slow gating of the heterodimer without MTSEA modification. (A) Singlechannel trace at −60 mV revealed two current levels and several short inactivation events. (B) Voltage activation experiment to examine slowgating transitions at the macroscopic current level (n = 3). (C) Temperaturejump 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) Slowgating behaviors of the MTSEAmodified heterodimers. (D) Singlechannel recording of a MTSEAmodified 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 slowgating transition rates, the schemes can be reduced to linear models. (B) Slowgate open probability contributes to the apparent MTSEAmodification rate. (Left) Calculated currentinduction 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 singleexponential 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) Currentinduction 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.
