A fluorescence probe of acetylcholine receptor conformation and local anesthetic binding

Abstract

The fluorescent dye ethidium bromide binds to the acetylcholine receptor with an apparent K d of 3 μ M and a stoichiometry of 1 molecule of ethidium per α-bungarotoxin site. Time dependent fluorescent increases were observed upon addition of carbamylcholine, the amplitude and half-time of which were dependent on the Carb 1 1 Abbreviations: AcCh, acetylcholine; AcChR, acetylcholine receptor; Carb, carbamylcholine; α-Bgt, α-bungarotoxin; 125I-α-Bgt, 125I-α-bungarotoxin; ANS, 1-anilino-8-naphthalene sulfonate. concentration. It appeared that these fluorescence increases resulted from a lowering of the K d for ethidium as the AcChR-Carb complex underwent an isomerization from low to high affinity form(s) for carb, and more ethidium was bound. Titration with the local anesthetic procaine led to ethidium fluorescence increases at low procaine concentrations, followed by a fluorescence decrease at higher procaine concentrations to that level induced by saturating α-bungarotoxin. Thus it appeared that the ethidium binding site either interacted with or was identical with local anesthetic binding site(s).