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Figure 2. Rate constants for the reaction of MTSEA with substituted Cys mutants in the absence (â¢) and presence (â) of ACh. When reagent was added with ACh, the ACh concentration was 10à the EC50 for the mutant. The protocols are given in methods. MTSEA concentrations ranged from 2.5 μM to 5 mM. Horizontal lines connect the mean rate constants in the two conditions. Each symbol is the mean of three to seven independent determinations. Thick lines represent the SEM where it extends beyond the symbol. The holding potential was â50 mV.
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Figure 3. Comparison of the rate constants for the reactions of AEAETS (diamonds), MTSEA (circles), MTSET (squares), and MTSEH (triangles) with substituted Cys mutants in the absence (filled symbols) and presence (open symbols) of ACh. Thick lines indicate the SEMs of the rate constants where larger than the symbol. The reactions of MTSET and MTSEH with αS248C, both in the presence and absence of ACh, were undetectable. For αL258C, the rate constant for the reaction with AEAETS in the absence of ACh is an upper limit because AEAETS induced some current. The holding potential was â50 mV. Each symbol is the average of two to seven independent measurements.
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Figure 4. Reaction of αT244C in the desensitized state. (A) Experimental protocol for determining the extent of MTSEA reaction with αT244C in the desensitized state. Applications of 60 μM ACh are indicated by downward arrows, of bath solution by upward arrows, and 85 μM MTSEA by horizontal bars. The time scale is given as the abscissa in B. Two initial responses elicited by 10-s applications of ACh were followed by a 4-min application of ACh, during which the current declined to 20% of its peak value by desensitization. Bath solution was applied for 15 s, and then MTSEA was applied for 30 s. After a 15-s wash, ACh was reapplied several times and the responses increased as the receptors recovered from desensitization. MTSEA was applied again for 30 s to the receptors in the resting state, and the effect of this application was assayed by a final application of ACh. The holding potential was â50 mV. (B) The inhibition, 1 â It/I0, due to both desensitization and the reaction of MTSEA is plotted as a function of the recording time, corresponding to the experiment in A. The recovery from desensitization was fit by a single exponential function with a time constant of 106 ± 27 s (n = 5), characteristic of the mutant αT244C.
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Figure 5. Rate constants for the reactions of thiosulfonate derivatives with αT244C in the absence of ACh as a function of membrane potential. Second-order rate constants for reactions with AEAETS (filled diamonds), MTSEA (filled circles), MTSEA at pH 6.5 (unfilled hexagons with dot), MTSET (filled squares), and MTSEH (filled triangles) are shown as a function of membrane potential. Nonlinear-least-squares fit of the data by Eq. 7 yielded the following parameters (parameters without errors were assumed): for MTSEA, C = 390 ± 55, D = 0, zδ = 0.05 ± 0.05; for AEAETS, C = 1.10 ± 0.06, D = 0, zδ = 0.040 ± 0.021; for MTSET, C = 1.28 ± 0.08, D = 0.01, zδ = 0.40 ± 0.02. These parameters were used to generate the curves. For MTSEH, for which z = 0, a line of zero slope was drawn at the mean κ.
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Figure 6. Rate constants for the reactions of Cys-substituted mutants in the presence of ACh with AEAETS (â), MTSEA (â), MTSET (â¡), and MTSEH (âµ) as a function of membrane potential. Means and SEM for three to seven determinations are shown. Nonlinear least squares fit of the data by Eq. 7 yielded the following parameters (parameters without errors were assumed), which were used to generate the curves: for αT244C and MTSEA, C = 46,900â± 4,190, D = 2, zδ = 0.38 ± 0.04; for αT244C and AEAETS, C = 21,200 ± 410, D = 0.007 ± 0.0004, zδ = 0.54 ± 0.009; for αT244C and MTSET, C = 307 ± 60, D = 0.01, zδ = 0.44 ± 0.06; for αS248C and MTSEA, C = 6.6 ± 1.8, D = 1, zδ = 0.28 ± 0.12; for αS248C and AEAETS, C = 11 ± 0.5, D = 0.007, zδ = 0.31 ± 0.02; for αL251C and MTSEA, C = 1,680 ± 16, D = 0.09 ± 0.003, zδ = 0.15; for αL251C and AEAETS, C = 6,060 ± 270, D = 0.007, zδ = 0.26 ± 0.01; for αL258C and AEAETS, C = 131 ± 130, D = 0.007, zδ = 0.12 ± 0.04. For αT244C and MTSEH, z = 0, and the overall mean of the mean κ at the three ÏM, 0.21 ± 0.05, is plotted with zero slope.
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Scheme I.
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Figure 7. Electrical distance, δ, from the extracellular medium to the probed Cys, determined in the presence of ACh. The δ were obtained from the fits in Fig. 6, assuming z = 1 for MTSET (â¡) and z = 2 for AEAETS (â).
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Figure 8. The relative rates of reaction and the intrinsic electrostatic potential in the channel at αT244C, αL251C, and αL258C. (A) The rate constants for the reactions of MTSEA (circles), AEAETS (diamonds), and MTSET (squares) with substituted Cys in αM2 were determined at various membrane potentials (Fig. 6), and the rate constants at zero membrane potential was determined by interpolation or extrapolation. Filled symbols are for reactions in the absence of ACh and open symbols are for reactions in the presence of ACh. These rate constants were divided by the rate constants for the reactions of MTSEH with the same substituted Cys, to obtain relative rates of reaction with the Cys in the channel. Also, the rate constants for the reactions of each of the charged thiosulfonates with 2-mercaptoethanol in solution was divided by the rate constant for the reaction of MTSEH with 2-mercaptoethanol to obtain relative rates of reaction with a simple thiol in solution. The relative rate constants for the reactions with receptor were divided by the relative rate constants for the reactions with 2-mercaptoethanol to obtain Ï0 for each mutant and reactant, both in the presence and absence of ACh. (B) The intrinsic potentials, ÏS, were calculated from Eq. 10. Symbols are as in A.
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Figure 9. Scheme of the electrostatics and the gate of the ACh receptor channel in the open and closed states. The sum of δÏM + ÏS is indicated by a contour plot through the channel with equipotentials every 25 mV. For the open state, δ is from Fig. 7, ÏM = â50 mV, ÏS is the average of the values at each residue in Fig. 8 B; for the closed state, δ = 0 and ÏS are the average of the values in Fig. 8 B. In the open state, δÏM + ÏS extrapolates to â¼â250 mV at αE241, the last contour shown.
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Scheme II.
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Figure 1. Determination of the rate constants for the reactions of thiosulfonates with substituted cysteines. (A–C) Reaction of AEAETS with αT244C in the presence of ACh. (A) Current recorded in the absence of ACh while clamping an oocyte expressing αT244C receptor to 25 mV for 1 s, −25 mV for 0.8 s, −75 mV for 0.4 s, and −50 mV for 0.25 s. (B) Currents induced by 60 μM ACh under the same voltage protocol as in A. (C) Currents in the presence of 5 μM AEAETS plus 60 μM ACh under the same voltage protocol as in A. Reagent and ACh were added before the start of the first voltage step. The time course of inhibition was approximately linear, and the pseudo-first order reaction rate constant at each voltage was estimated by the slope divided by the current amplitude at the beginning of each voltage interval. Linear fits and slope values are shown for the reactions at −25 and −100 mV. The records in A–C were obtained from the same cell. (D–E) Reaction of AEAETS with αT244C in the absence of ACh. (D) ACh-induced currents before and after applications of 1 mM AEAETS in the absence of ACh. The following sequence of applications was repeated five times: 60 μM ACh for 10 s, wash 4 min, AEAETS for 2–4 min (beginning at arrows), and wash 3 min; ACh was applied for 10 s to obtain the final response. The cumulative duration of exposure to AEAETS before each ACh-induced response is indicated next to the peak of current. The clamp potential was −50 mV. (E) Peak ACh-induced currents in D (○), normalized to the initial ACh- induced current, plotted against the cumulative time of exposure to AEAETS. The solid line is a single exponential fit (see methods). (F–G) Reaction of 10 mM AEAETS with αS248C in the presence of ACh. (F) An oocyte was alternatively exposed to 25 μM ACh in the absence and presence of AEAETS and allowed to recover for 4 min after each application. The following sequence of applications was repeated seven times: ACh for 10 s, wash 4 min, ACh plus AEAETS for 2–20 s (beginning at arrows), and wash 4 min. ACh for 10 s was added again at the end. The clamp potential was −50 mV. The cumulative duration of exposure to AEAETS before each ACh-induced response is indicated next to the peak of current. (G) As in E, normalized peak currents were plotted against the cumulative duration of exposure to AEAETS (○).
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