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Figure 1. Schematic representation of the muscle-type ACh receptor. (A) The receptor complex in the membrane. (B) The common membrane topology of its subunits. (C) The aligned sequences of four mouse-muscle receptor subunits at the cytoplasmic ends of their M2 membrane-spanning segments and the M1–M2 loops. The sequences in C are from the region of the subunits covered with the shaded circle in B. The residues that were mutated in the work described here are in bold type. The numbering is that of the mature sequences.
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Figure 2. Responses of complexes of subunits mutated in the Glu ring. Oocytes were injected with all four types of subunits. In all cases, α contained the mutation T244C; this is designated α− if E241 is not also mutated, and α0 if in addition E241 is mutated to Q (i.e., the superscript indicates the charge of the residue in the position of the Glu ring). Wild-type β is designated β−; β with the mutation E252 to Q is designated β0; and β with the mutation E252 to K is designated β+. Wild-type γ is designated γ0. Wild-type δ is designated δ−, and δ with the mutation E255 to Q is designated δ0. The complexes tested were: α−2β−γ0δ− (i.e., a pseudo wild type with only αT244 mutated to C; ring charge, −4; square); α−2β0γ0δ− (ring charge, -3; up triangle); α−2β−γ0δ0 (ring charge, −3; down triangle); α02β−γ0δ− (ring charge, −2; diamond); α−2β+γ0δ− (ring charge, −2; hexagon); and α02β0γ0δ− (ring charge, −1; a circle). (A) The average maximum current (−IMAX) obtained from the fit of the Hill equation to the responses at various concentrations of ACh. (B) The average EC50 obtained from the fit of the Hill equation. The least-squares linear fit to the log(EC50) is shown. In each case, the abscissa is the sum of the charges in the ring. The numbers of independent experiments are indicated next to the symbols. The bars represent the standard errors of the means.
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Figure 3. Kinetics of the reaction of MTSEA with the Cys at α244 exemplified with α02β0γ0δ−. MTSEA was applied to oocytes expressing α02β0γ0δ− in both the closed (A and B) and open (C and D) states at a holding potential of −50 mV. The sequence of applications to the oocytes was: 200 μM ACh for 10 s; bath solution for 3 min; 5 mM MTSEA for 2–16 s (beginning at arrows) for the closed state (A) or 50 μM MTSEA together with 200 μM ACh for 5–20 s (arrows) for the open state (C); bath solution for 3 min. This sequence was repeated several times, resulting in cumulative applications of MTSEA for the times shown under the peaks of the test responses to ACh. There was no decline in current during the 10-s applications of ACh; the return of the current to baseline began with the wash out of ACh. The peak current was normalized and plotted against the cumulative MTSEA exposure time in the absence (B) and presence (D) of ACh. Solid lines indicate least squares single exponential fits to the data, which yield the pseudo first- and second-order rate constants for the reactions (see materials and methods).
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Figure 4. Second-order rate constants for the reactions of thiosulfonate reagents with α02β0γ0δ− as a function of the transmembrane potential. The rate constants were determined as in Fig. 2 for MTSEH (triangle), MTSEA (circle), and AEAETS (diamond) in the absence (filled symbols) and presence (unfilled symbols) of ACh, at various holding potentials. In each case, the mean ± SEM is plotted for recordings from three to four cells.
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Figure 5. The second-order rate constants for the reaction of thiosulfonate reagents with receptors bearing different ring charges. The rate constants for the reactions at zero transmembrane potential are plotted for MTSEH (A), MTSEA (B), and AEAETS (C) applied both in the absence (filled symbols) and presence (unfilled symbols) of ACh. The correspondence between symbols and subunit combinations is the same as in Fig. 2. The lines are the linear least-squares fits.
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Figure 7. ρ0 (A) and ΔΔG 0 and zψS (B) for AEAETS and MTSEH. The details are as in Fig. 6.
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Figure 6. ρ0 and ΔΔG 0 for MTSEA and MTSEH as a function of ring charge. (A) The ratio of the rate constants for the reactions with the receptor was divided by the ratio of the rate constants for the reactions of MTSEA and of MTSEH with 2-mercaptoethanol in solution, thereby normalizing for the difference in the intrinsic reactivities of the two reagents (Pascual and Karlin 1998). This ratio of ratios, ρ0, is plotted versus the ring charge. Rate constants for the reactions of the mutant receptors in the presence (unfilled symbols) and absence (filled symbols) of ACh, at zero transmembrane potential, are from the data in Fig. 5. The symbols correspond to the same combinations of subunits as in Fig. 2. (B) ΔΔG 0 was calculated as −RTln(ρ0) and zψS was calculated as ΔΔG 0/F.
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Figure 8. A simple model of the open channel. A cylindrical pore of 8-Å diameter and 30-Å length crosses a slab representing the bilayer and the bilayer-embedded receptor. Water is on either side of the slab and within the channel (dark fill). The water has a dielectric constant of 80, and the slab (no fill) has a dielectric constant of 2. Measuring along the long axis (x axis) of the channel from its midpoint, the intracellular end is at x = −15 Å and the extracellular end is at x = 15 Å. Point charges are placed at the vertices of a pentagon at x = −14 Å and at a distance from the x axis (r coordinate) of 5 Å. A model of MTSEA is shown in the channel with the positively charged ammonium centered at r = 0 Å, x = −14 Å. The point charges were given values of −1 or 0. The closed channel includes as a gate, a disc of dielectric constant 2, 8-Å diameter and 2-Å thick, from x = −12 to −14 Å. EX, extracellular; IN, intracellular; MEM, membrane.
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Figure 9. Electrostatic free energies of transfer into the model channel. (A). The differences (ΔΔG 0) in the ΔG 0 of MTSEA and MTSEH (circles) and of AEAETS and MTSEH (diamonds) are shown for both the open (unfilled symbols) and closed (filled symbols) states. In the open state, MTSEA was oriented as in Fig. 8, with the ammonium N at x = −14 Å, r = 0 Å. The ammonium of AEAETS closest to the -S− was placed at r = 0 Å and x = −14 Å, and the second ammonium closest to the -SO2− was at r = 0 Å and x = −4.4 Å. The hydroxyl hydrogen of MTSEH was placed in the same location as the ammonium of MTSEA, and the orientation of the molecule was similar. In the closed state, the head groups of the reagents were moved to x = −10 Å, while the orientations were unchanged. (B). ΔG 0 for the transfer of a Na+ from the extracellular solution to the channel was calculated at 1-Å intervals, from x = −18 to 18 Å. (The channel extends from −15 to 15 Å.) In the closed state, the Na+ was excluded from the gate, which extends from x = −14 to −12 Å (double-headed arrow).
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