Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: IV. Quinacrine: a fluorescent probe for the conformational transitions of the cholinergic receptor protein in its membrane-bound state

Quinacrine, like a typical local anaesthetic, blocks the response of Electrophorus electricus electroplaque in vivo in a non-competitive manner and enhances, in vitro, the affinity of the cholinergic receptor present in Torpedo marmorata membrane fragments for acetylcholine. The interaction of quinacrine with T. marmorata membrane fragments can be followed by differential fluorescence spectroscopy either upon direct illumination (λ_Ex = 350 nm) or by energy transfer from membrane proteins (λ_Ex = 290 nm). Carbamylcholine and most of the cholinergic ligands tested cause an increase of the light intensity emitted by membrane-bound quinacrine under conditions of direct excitation; all these effects are blocked by a preincubation of the membrane fragments with the α-toxin from Naja nigricollis. When quinacrine is excited by energy transfer, carbamylcholine, phenyltrimethylammonium and hexamethonium cause an increase of fluorescence but flaxedil, tetraethylammonium and the α-toxin give a much smaller fluorescence increase or none.Local anaesthetics like prilocaine or quotane cause a decrease of fluorescence intensity of membrane-bound quinacrine in both the presence and absence of carbamylcholine. Quantitative studies on quinacrine binding and fluorescence as a function of quinacrine concentration reveal at least two populations (saturable and non-saturable) of binding sites, the saturable one being identical or closely related to the specific site of action of local anaesthetics. It is concluded that binding of cholinergic ligands primarily increases the quantum yield of a fraction of bound quinacrine.The curves of variation of fluorescence intensity with agonist and antagonist concentrations determined under conditions of direct illumination, closely resemble the binding curves determined at equilibrium with radioactive ligands. Under these conditions quinacrine therefore enables us to determine the occupancy of the receptor site by cholinergic ligands. On the other hand, the change of quinacrine fluorescence observed by energy transfer, which takes place with some of the cholinergic ligands but not with others, and does not correlate with any variation of the intrinsic fluorescence of membrane proteins, most likely reflects a change of structure bearing a qualitative relationship to the pharmacological activity of the tested ligands.