Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 1. Equilibrium studies

Abstract

The interactions between the fluorescent probe ethidium and acetylcholine receptor enriched membranes from Torpedo californica [electroplax] are described. One class of saturable ethidium sites was blocked by .alpha.-bungarotoxin and therefore reflects direct binding to the receptor (Kd .apprx. 3 .mu.M; stoichiometry-1 ethidium site/2 .alpha.-bungarotoxin sites). The 2nd class of sites was nonsaturable and unaffected by .alpha.-toxin and was therefore considered nonspecific in nature. The increase in fluorescence intensity observed upon addition of cholinergic agonists and antagonists accurately reflects the Kd and stoichiometry of the high-affinity receptor sites for these ligands. The effects of local anesthetics are complex in nature and depend on the structure of the ligand. For carbamylcholine, the increase in fluorescence intensity was due to an increase in the quantum yield of the dye bound to the membrane rather than a dye uptake. In general, ethidium does not appear to strongly alter the properties of the membrane-bound acetylcholine receptor and can therefore be profitably used as a spectroscopic probe.