Independent activation of the acetylcholine receptor from Torpedo californica at two sites

Abstract

Membrane vesicles enriched in acetylcholine receptor were prepared from the electroplax tissue of Torpedo californica. The receptor was reduced with dithiothreitol to expose a sulfhydryl group near the ligand binidng site and then treated in one of the following ways: (1) affinity alkylated treated in one of the following ways: (1) affinity alkylated with bromoacetylcholine, a receptor activator, (2) affinity alkylated with maleimidobenzyltrimethylammonium, a receptor inhibitor, or (3) reoxidized to the native state with dithiobis(2-nitrobenzoate). The affinity labels blocked half of the binding sites for alpha-bungarotoxin. The toxin sites not protected by the affinity labels were protected by carbamylcholine based on studies of toxin binding kinetics. The functional response of native and affinity-alkylated receptors was measured by a sodium ion flux procedure. In the absence of added cholinergic activators, only slow ion flux was observed. In the presence of carbamylcholine, a receptor activator, both native and modified membranes showed the increased sodium flux associated with functional receptors. The concentration of carbamylcholine required for a 50% maximal response was higher in the affinity-labeled membranes. Preincubation of the membranes with carbamylcholine blocked the increased ion flux, indicating that desensitization could be induced. The results provide evidence for the existence of two functional sites on the acetylcholine receptor. Each site corresponds to a bungarotoxin binding site and can be independently activated and desensitized.

Functional architecture of the nicotinic acetylcholine receptor: A prototype of ligand-gated ion channels

“…They differ by their kinetics of binding and dissociation of a bungarotoxin (Weber & Changeux 1974a, b, c;Maelicke & Reich, 1976;Maelicke et al, 1977;Kang & Maelicke, 1980;Maelicke et al, 1989), by their affinities for the competitive antagonists d-tubocurarine (Neubig & Cohen, 1979;Pedersen & Cohen, 1990a) or decamethonium and by their different labeling sensitivity to affinity reagents such as bromoacetylcholine, MBTA (Karlin et al, 1976;Damle & Karlin, 1978;Delegeane & McNamee, 1980;Ratnam et al, 1986) or lophotoxin (Culver, Fenical & Taylor, 1984). Also some antibodies differentially block the binding of oL bungarotoxin to one or the other a subunit (Watters & Maelicke, 1983;Gu, Silberstein & Hall, 1985;Whiting et al, 1985;Dowding & Hall, 1987).…”

The Molecular Neurobiology of the Acetylcholine Receptor

“…Although these sites may not be identical in structure, both are involved in channel opening. Even when one site is blocked by MBTA, ion flux could be induced by agonist binding to the second high-affinity site (Delegeane & McNamee 1980). ACh binding to these sites was shown to be weakly cooperative under desensitizing conditions (Fels et al 1982); models involving initially nonequivalent sites characterized by concave Scatchard plots (Prinz Maelicke 1983) and long-lived, variable affinity states (Chang et al 1984) have been proposed.…”

Acetylcholine receptor kinetics

“…Though only one of these sites can be affinity labeled after disulfide reduction, both sites appear capable of triggering permeability and desensitization response (Delegeane & McNamee, 1980). Perhaps the most careful recent measurements suggest that there are two ACh binding sites corresponding to the two toxin binding sites per monomer.…”