DELIMITING THE BINDING-SITE FOR QUATERNARY AMMONIUM LIDOCAINE DERIVATIVES IN THE ACETYLCHOLINE-RECEPTOR CHANNEL

The triethylammonium QX-314 and the trimethylammonium QX-222 are lidoc aine derivatives that act as open-channel blockers of the acetylcholin e (ACh) receptor. When bound, these blockers should occlude some of th e residues lining the channel. Eight residues in the second membrane-s panning segment (M2) of the mouse-muscle alpha subunit were mutated on e at a time to cysteine and expressed together with wild-type beta, ga mma, and delta subunits in Xenopus oocytes. The rate constant for the reaction of each substituted cysteine with 2-aminoethyl methanethiosul fonate (MTSEA) was determined from the time course of the irreversible effect of MTSEA on die ACh-induced current. The reactions were carrie d out in the presence and absence of ACh and in the presence and absen ce 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 substitut ed for residues from alpha Val255, one third of the distance in from t he extracellular end of M2, to alpha Glu241, flanking the intracellula r end of M2, but not with cysteine substituted far alpha Leu258 or alp ha Glu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for alpha Leu258 and alpha Glu262 were consider ably faster in the presence of ACh than in its absence. That QX-314 an d QX-222 did not protect alpha L258C and alpha 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-Angstrom ov erall length of the blockers and the alpha-helical conformation of M2 in the open state, the binding site for both blockers extends from alp ha Val255 down to alpha Ser248.