Wilson GG et al. (2000), The intrinsic electrostatic potential and the i...

XB-ART-11565

J Gen Physiol 2000 Feb 01;1152:93-106.

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The intrinsic electrostatic potential and the intermediate ring of charge in the acetylcholine receptor channel.

Wilson GG , Pascual JM , Brooijmans N , Murray D , Karlin A .

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A ring of aligned glutamate residues named the intermediate ring of charge surrounds the intracellular end of the acetylcholine receptor channel and dominates cation conduction (Imoto et al. 1988). Four of the five subunits in mouse-muscle acetylcholine receptor contribute a glutamate to the ring. These glutamates were mutated to glutamine or lysine, and combinations of mutant and native subunits, yielding net ring charges of -1 to -4, were expressed in Xenopus laevis oocytes. In all complexes, the alpha subunit contained a Cys substituted for alphaThr244, three residues away from the ring glutamate alphaGlu241. The rate constants for the reactions of alphaThr244Cys with the neutral 2-hydroxyethyl-methanethiosulfonate, the positively charged 2-ammonioethyl-methanethiosulfonate, and the doubly positively charged 2-ammonioethyl-2'-ammonioethanethiosulfonate were determined from the rates of irreversible inhibition of the responses to acetylcholine. The reagents were added in the presence and absence of acetylcholine and at various transmembrane potentials, and the rate constants were extrapolated to zero transmembrane potential. The intrinsic electrostatic potential in the channel in the vicinity of the ring of charge was estimated from the ratios of the rate constants of differently charged reagents. In the acetylcholine-induced open state, this potential was -230 mV with four glutamates in the ring and increased linearly towards 0 mV by +57 mV for each negative charge removed from the ring. Thus, the intrinsic electrostatic potential in the narrow, intracellular end of the open channel is almost entirely due to the intermediate ring of charge and is strongly correlated with alkali-metal-ion conductance through the channel. The intrinsic electrostatic potential in the closed state of the channel was more positive than in the open state at all values of the ring charge. These electrostatic properties were simulated by theoretical calculations based on a simplified model of the channel.

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Species referenced: Xenopus laevis
Genes referenced: tbx2

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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 M1âM2 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.
	Figure 2. Responses of complexes of subunits mutated in the Glu ring. Oocytes were injected with all four types of subunits. In all cases, Î± contained the mutation T244C; this is designated Î±â if E241 is not also mutated, and Î±0 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 Î² is designated Î²â; Î² with the mutation E252 to Q is designated Î²0; and Î² with the mutation E252 to K is designated Î²+. Wild-type Î³ is designated Î³0. Wild-type Î´ is designated Î´â, and Î´ with the mutation E255 to Q is designated Î´0. The complexes tested were: Î±â2Î²âÎ³0Î´â (i.e., a pseudo wild type with only Î±T244 mutated to C; ring charge, â4; square); Î±â2Î²0Î³0Î´â (ring charge, -3; up triangle); Î±â2Î²âÎ³0Î´0 (ring charge, â3; down triangle); Î±02Î²âÎ³0Î´â (ring charge, â2; diamond); Î±â2Î²+Î³0Î´â (ring charge, â2; hexagon); and Î±02Î²0Î³0Î´â (ring charge, â1; a circle). (A) The average maximum current (âIMAX) 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.
	Figure 3. Kinetics of the reaction of MTSEA with the Cys at Î±244 exemplified with Î±02Î²0Î³0Î´â. MTSEA was applied to oocytes expressing Î±02Î²0Î³0Î´â in both the closed (A and B) and open (C and D) states at a holding potential of â50 mV. The sequence of applications to the oocytes was: 200 Î¼M ACh for 10 s; bath solution for 3 min; 5 mM MTSEA for 2â16 s (beginning at arrows) for the closed state (A) or 50 Î¼M MTSEA together with 200 Î¼M ACh for 5â20 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).
	Figure 4. Second-order rate constants for the reactions of thiosulfonate reagents with Î±02Î²0Î³0Î´â 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 Â± SEM is plotted for recordings from three to four cells.
	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.
	Figure 7. Ï0 (A) and ÎÎG 0 and zÏS (B) for AEAETS and MTSEH. The details are as in Fig. 6.
	Figure 6. Ï0 and ÎÎG 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, Ï0, 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) ÎÎG 0 was calculated as âRTln(Ï0) and zÏS was calculated as ÎÎG 0/F.
	Figure 8. A simple model of the open channel. A cylindrical pore of 8-Ã diameter and 30-Ã 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 = â15 Ã and the extracellular end is at x = 15 Ã. Point charges are placed at the vertices of a pentagon at x = â14 Ã and at a distance from the x axis (r coordinate) of 5 Ã. A model of MTSEA is shown in the channel with the positively charged ammonium centered at r = 0 Ã, x = â14 Ã. The point charges were given values of â1 or 0. The closed channel includes as a gate, a disc of dielectric constant 2, 8-Ã diameter and 2-Ã thick, from x = â12 to â14 Ã. EX, extracellular; IN, intracellular; MEM, membrane.
	Figure 9. Electrostatic free energies of transfer into the model channel. (A). The differences (ÎÎG 0) in the ÎG 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 = â14 Ã, r = 0 Ã. The ammonium of AEAETS closest to the -Sâ was placed at r = 0 Ã and x = â14 Ã, and the second ammonium closest to the -SO2â was at r = 0 Ã and x = â4.4 Ã. 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 = â10 Ã, while the orientations were unchanged. (B). ÎG 0 for the transfer of a Na+ from the extracellular solution to the channel was calculated at 1-Ã intervals, from x = â18 to 18 Ã. (The channel extends from â15 to 15 Ã.) In the closed state, the Na+ was excluded from the gate, which extends from x = â14 to â12 Ã (double-headed arrow).

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