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Original Article

^a From the Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032
^b From the Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York 10032
^c From the Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032
^d From the Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032
Center for Molecular Recognition, Columbia University, 630 West 168th Street, New York, NY 10032.212-305-5594

ak12{at}columbia.edu

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 subunit contained a Cys substituted for Thr244, three residues away from the ring glutamate Glu241. The rate constants for the reactions of Thr244Cys 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.

Key Words: nicotinic • mutagenesis • reaction kinetics • conductance • selectivity

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