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Article

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

From the ^* Center for Molecular Recognition, Department of Neurology, Department of Physiology and Cellular Biophysics, and ^|| Department of Biochemistry and Molecular Biophysics, Columbia University, New York 10032

The triethylammonium QX-314 and the trimethylammonium QX-222 are lidocaine derivatives that act as open-channel blockers of the acetylcholine (ACh) receptor. When bound, these blockers should occlude some of the residues lining the channel. Eight residues in the second membrane-spanning segment (M2) of the mouse-muscle subunit were mutated one at a time to cysteine and expressed together with wild-type β, , and subunits in Xenopus oocytes. The rate constant for the reaction of each substituted cysteine with 2-aminoethyl methanethiosulfonate (MTSEA) was determined from the time course of the irreversible effect of MTSEA on the ACh-induced current. The reactions were carried out in the presence and absence of ACh and in the presence and absence of QX-314 and QX-222. These blockers had no effect on the reactions in the absence of ACh. In the presence of ACh, both blockers retarded the reaction of extracellularly applied MTSEA with cysteine substituted for residues from Val255, one third of the distance in from the extracellular end of M2, to Glu241, flanking the intracellular end of M2, but not with cysteine substituted for Leu258 or Glu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for Leu258 and Glu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect L258C and E262C against reaction with MTSEA in the presence of ACh implies that protection of the other residues was due to occlusion of the channel and not to the promotion of a less reactive state from a remote site. Given the 12-Å overall length of the blockers and the -helical conformation of M2 in the open state, the binding site for both blockers extends from Val255 down to Ser248.

Key Words: cysteine mutagenesis • reaction kinetics • methanethiosulfonate • open-channel block

Abbreviations: ACh, acetylcholine; M2, second membrane-spanning segment; MTSEA, 2-aminoethyl methanethiosulfonate; MTSEH, 2-hydroxyethyl ethanethiosulfonate; NCI, noncompetitive inhibitor

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