We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine (D-eserine) with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmoruta electric tissue by means of ligandinduced ion flux into nAChR-rich membrane vesicles and of equilibrium binding. We find that (-)physostigmine induces cation flux (and also binds to the receptor) even in the presence of saturating concentrations of antagonists of acetylcholine, such as D-tubocurarine, a-bungarotoxin or antibody WF6. The direct action on the acetylcholine receptor is not affected by removal of the methylcarbamate function from the drug and thus is not due to carbamylation of the receptor. Antibodies FK1 and benzoquinonium antagonize channel activation (and binding) of eserine, suggesting that the eserine binding site(s) is separate from, but adjacent to, the acetylcholine binding site at the receptor. In addition to the channel activating site(s) with an affinity of binding in the 50 pM range, there exists a further class of low-affinity (Kd -mM) sites from which eserine acts as a direct blocker of the acetylcholine-activated channel. Our results suggest the existence of a second pathway of activation of the nAChR channel.The nicotinic acetylcholine receptor (nAChR) from muscle, brain and electric tissue is a ligand-gated cation channel [l -31. Binding of acetylcholine, or its agonists, induces transient openings of the channel. Antagonists compete with acetylcholine and its agonists in binding to the receptor thereby blocking the channel-activating action of agonists. Other ligands (non-competitive blockers, direct channel blockers) modulate the agonist-activated channel by binding to separate sites [l, 31. Physostigmine (eserine) is a slowly reversible inhibitor of acetylcholine esterase which acts by carbamylation of the active serine residue within the 'esteratic site' of the enzyme 141. action on the receptor might at least equal in importance the action on the enzyme. At higher concentrations, eserine exhibits noncompetitive antagonism with respect to cholinergic agonists, accompanied by rapid opening and closing events (flickering) of the activated channels [8 -10, 141. Vesicular membrane fragments from Torpedo electric tissue permit the direct measurement of both ligand binding to the nAChR and ligand-induced ion flux through the nAChRintegral cation channel. To remain within the time range of excitatory events at the neuromuscular junction, ion flux is best studied by time-resolved fluorimetry employing rapidmixing devices [15][16][17][18]. For this purpose, we prepared sucrose-gradient-purified membrane vesicles from T. marmoruta electric tissue [19], loaded them with 1, 3,6,8-pyrene tetrasulfonate [20], and monitored the quenching of fluorescence induced by influx of Cs' [21] after rapid mixing, in a stoppedflow apparatus, of dye-loaded membrane vesicles with a buffer containing the heavy metal quencher and eserine. In addition to thereby establishing an agonistic action of eserine at Torpedo nAChR, o...

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We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine and several structurally related compounds with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles, direct binding studies and photoaffinity labeling. (-)Physostigmine acts as a channel-activating ligand at low concentrations and as a direct channel blocker at elevated concentrations. Channel activation is not inhibited by desensitizing concentrations of ACh or ACh-competitive ligands (including alpha-bungarotoxin and D-tubocurarine) but is inhibited by antibody FK1 and several other compounds. From photoaffinity labeling using tritiated physostigmine and mapping of the epitope for the Phy-competitive antibody FK1, the binding site for physostigmine is located within the alpha-subunit of the Torpedo nAChR and is distinct from the acetylcholine binding site. Our data suggest a second pathway of nAChR channel activation that may function physiologically as an allosteric control of receptor activity.