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Article

Inferences from Rates of Reaction of Thiosulfonates with Substituted Cysteines in the M2 Segment of the Subunit

Juan M. Pascual^*, and Arthur Karlin^*

From the ^* Center for Molecular Recognition, and Division of Pediatric Neurology, College of Physicians and Surgeons, Columbia University, New York 10032

Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl-specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for Thr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with T244C, L251C, and L258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of M2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than Thr244.

Key Words: conductance • gate • ion selectivity • reaction kinetics • sulfhydryl

Abbreviations: ACh, acetylcholine; AEAETS, 2-aminoethyl-2-aminoethanethiosulfonate; 2-ME, 2-mercaptoethanol; MTS, methanethiosulfonate; MTSEA, 2-aminoethyl-MTS; MTSEH, 2-hydroxyethyl-MTS; MTSET, 2-trimethylammonioethyl-MTS

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