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

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 exp ressed in Xenopus laevis oocytes. In all complexes, the alpha subunit conta ined a Cys substituted for alpha Thr244, thr-ee residues away from the ring glutamate alpha Glu241. The rate constants for the reactions of alpha Thr2 44Cys with the neutral 2-hydroxyethyl-methanethiosulfonate, the positively charged 2-ammonioethyl-methanethiosulfonate, and the doubly positively char ged 2-ammonioethyl-2'-ammonioethanethiosulfonate were determined from the r ates of irreversible inhibition of the responses to acetylcholine. The reag ents were added in the presence and absence of acetylcholine and at various transmembrane potentials, and the e-ate constants were extrapolated to zer o transmembrane potential. The intrinsic electrostatic potential in the cha nnel in the vicinity of the ring of charge was estimated from the ratios of the rate constants of differ-entry 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-io n conductance through the channel. The intrinsic electrostatic potential in the closed state of the channel was more positive than in the open state a t all values of the ring charge. These electrostatic properties were simula ted by theoretical calculations based on a simplified model of the channel.