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 α subunit contained a Cys substituted for αThr244, three residues away from the ring glutamate αGlu241. The rate constants for the reactions of αThr244Cys 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.
The Intrinsic Electrostatic Potential and the Intermediate Ring of Charge in the Acetylcholine Receptor Channel
Although the accessibilities of these residues from the two sides of the membrane in the open and closed states of the channel are known (Wilson and Karlin 1998), their distances from the cytoplasmic face of the phospholipid bilayer are not known (see Miyazawa et al. 1999).
Abbreviations used in this paper: ACh, acetylcholine; AEAETS, 2-ammonioethyl-2′-ammonioethanethiosulfonate dichloride; MTSEA, 2-ammonioethyl-methanethiosulfonate bromide; MTSEH, 2-hydroxyethyl-methanethiosulfonate.
Because ψDielectric depends on z, 0.5 zF ψDielectric is proportional to 0.5 z2, and only if ψFixed >> ψDielectric is ΔΔG0 proportional to the first power of the reagent charge, z.
The receptor exists in at least four different functional states, closed, open, and fast- and slow-desensitized. Only in the open state is the channel conducting. As discussed previously (Pascual and Karlin 1998), reagents added in the absence of ACh react predominantly with the closed state, and during brief (≤20 s) application of reagent together with ACh the reaction is with a mixture of receptors in the open state and with receptors in the fast-desensitized state. In the case of reactions with αT244C, the rate constant for the reaction of MTSEA with receptor that has been driven into the slow-desensitized state is two to three orders of magnitude smaller than with receptor in the mixed open and fast-desensitized states (our unpublished results). Furthermore, the dependence on transmembrane potential of the rate of reaction of αT244C with AEAETS in the presence of ACh suggests that the reaction is predominantly with receptor in the conducting open state, and not with receptor in the closed state or in either of the nonconducting desensitized states. For convenience, we call the mixed state within the first 20 s of ACh application the open state.
In the future, we will explore the theoretical implications of more subtle gate structures, of a more realistic lumen geometry, of moving the intermediate ring, of including the inner and outer rings of charges and the ring of lysines, and of varying the orientation of the reagents in the channel.
Even if the ammonium group of MTSEA were representative of a permeant cation and the hydroxyl group of MTSEH were representative of a water molecule displaced by the cation, ΔΔG0 does not include the entropy of transfer of the cation from bulk solution to the site, the value of which is unknown. The calculated electrostatic contribution to ΔG0 for the transfer of Na+ from bulk solution to the site is the electrostatic contribution to the enthalpy of transfer. It does not include van der Waals contributions to the enthalpy of transfer and does not include the entropy of transfer.
Gary G. Wilson, Juan M. Pascual, Natasja Brooijmans, Diana Murray, Arthur Karlin; The Intrinsic Electrostatic Potential and the Intermediate Ring of Charge in the Acetylcholine Receptor Channel. J Gen Physiol 1 February 2000; 115 (2): 93–106. doi: https://doi.org/10.1085/jgp.115.2.93
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