A second pathway of activation of the Torpedo acetylcholine receptor channel

Abstract

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...

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

“…Because neither etomidate nor azietomidate potentiates responses of the Torpedo nAChR, we did not begin this work expecting to find that TDBzl-etomidate acts as a potentiator. Interestingly the structure of TDBzl-etomidate, a substituted (1-phenylethyl-5-carboxylate)imidazole, has similarities to the substituted (2-amino-5-keto)thiazoles recently introduced as potentiators of several neuronal, but not muscle, nAChRs (42), but it is quite distinct from previously described allosteric modulators of muscle-type nAChRs such as physostigmine or galantamine, which activate via a site distinct from the agonist site (17,19,22). Although studies using higher resolution electrophysiological techniques will be necessary to determine the gating parameters altered by TDBzl-etomidate, the observed potentiation could result from a decrease of ACh K app from 25 to 10 M because the ACh response was characterized by a K app of 25 M and a Hill coefficient of ϳ1.…”

“…In contrast, most general anesthetics act as negative allosteric modulators of nAChRs (14). Positive allosteric nAChR modulators have been identified, including natural products such as physostigmine and galantamine, which are active on muscle and neuronal nAChRs (17)(18)(19), and compounds identified recently through high throughput drug screens that have selectivity for one or more neuronal nAChR subtypes (for reviews, see Refs. 20 and 21).…”

Barbiturate interactions at the human GABAA receptor: dependence on receptor subunit combination

“…Recent mutagenesis experiments addressing the GABA binding site demonstrate that mutants with up to 900 fold lower affinity for GABA are unchanged in their sensitivity for direct activation by pentobarbitone (Amin & Weiss, 1993), also suggesting a unique binding site for direct activation by pentobarbitone. This is comparable to another ligand gated ion channel, the nicotinic acetylcholine receptor, which is activated by physostigmine via a site separate from the acetylcholine binding site (Okonjo et al, 1991;Lena & Changeux, 1993). Krishek & Smart (1995) (Akaike et al, 1987;Peters, 1988;Robertson, 1989) have reported on the washout phenomenon observed with high concentrations of pentobarbitone.…”

Unconventional ligands and modulators of nicotinic receptors

“…The discovery of nicotinic APLs, i.e., ligands that can bind to and potentiate the activation of nAChRs by classical agonists, dates back to the 1980s, when studies from our laboratories demonstrated that the cholinesterase inhibitor physostigmine can interact with muscle nAChRs and activate single-channel currents in muscle fibers under cellattached configuration (Shaw et al, 1985;Sherby et al, 1985). Subsequently, physostigmine was found to activate Torpedo nAChRs as well as neuronal nAChRs expressed natively in hippocampal neurons or ectopically in fibroblasts (Okonjo et al, 1991;Pereira et al, 1993Pereira et al, , 1994. Physostigmine-induced activation of nAChRs was insensitive to blockade by competitive nAChR antagonists and could be observed when the receptors were desensitized by high agonist concentrations (Okonjo et al, 1991;Pereira et al, 1993;Schrattenholz et al, 1993).…”

“…Subsequently, physostigmine was found to activate Torpedo nAChRs as well as neuronal nAChRs expressed natively in hippocampal neurons or ectopically in fibroblasts (Okonjo et al, 1991;Pereira et al, 1993Pereira et al, , 1994. Physostigmine-induced activation of nAChRs was insensitive to blockade by competitive nAChR antagonists and could be observed when the receptors were desensitized by high agonist concentrations (Okonjo et al, 1991;Pereira et al, 1993;Schrattenholz et al, 1993). The monoclonal antibody raised against the Torpedo nAChR ␣ subunit and referred to as FK1 is the only known antagonist of the nicotinic agonistic action of physostigmine.…”