Arrangement of the subunits of the nicotinic acetylcholine receptor of Torpedo californica as determined by alpha-neurotoxin cross-linking.
[3H]Methyl-alpha-neurotoxin prereacted with dithiobis(succinimidyl propionate) (DTSP) can be covalently linked to each of the subunits of the nicotinic acetylcholine receptor in membranes from the electric tissue of Torpedo californica. Pronounced changes in the cross-linking pattern are observed upon prior incubation with receptor specific ligands and upon reduction and/or alkylation of the receptor. d-Tubocurarine has been shown to bind to two different sites in receptor-rich membranes. These sites are present in equal numbers but have different affinities [Neubig, R. R., & Cohen, J. B. (1979) Biochemistry 18, 5464-5475; Sine, S., & Taylor, P. (1981) J. Biol. Chem. 256, 6692-6699]. Using d-tubocurarine inhibition of [3H]-methyl-alpha-neurotoxin binding, we demonstrate two inhibitory constants for d-tubocurarine of 67 +/- 21 nM and 4.9 +/- 1.7 microM in unreduced membranes. We utilize the large difference in Ki's to preferentially block toxin cross-linking at the high affinity site for d-tubocurarine. Low concentrations of this competitive antagonist selectively block the cross-linking of toxin to the beta and gamma subunits of the receptor, suggesting that these subunits are located close to the toxin binding site which is also the high-affinity binding site for d-tubocurarine. Reduction of disulfide bonds alters the affinity of the receptor for alpha-neurotoxin. Alterations are also seen in the cross-linking pattern of DTSP-activated [3H]methyl-alpha-neurotoxin to reduced and alkylated membranes in the presence of tubocurarine. The constants for d-tubocurarine inhibition of [3H]methyl-alpha-neurotoxin binding to reduced and alkylated membranes are 172 +/- 52 nM and 2.4 +/- 0.4 microM. The effects of bromoacetylcholine, carbamoylcholine, gallamine, and procaine on the cross-linking pattern are also examined. Our observations are consistent with an arrangement of the subunits in the membrane of alpha beta alpha gamma delta.
D000478:Alkylation; D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D046911:Macromolecular Substances; D009498:Neurotoxins / Q000378:metabolism*; D010084:Oxidation-Reduction; D011950:Receptors, Cholinergic / Q000032:analysis*; D030162:Reptilian Proteins; D012910:Snake Venoms; D013388:Succinimides / Q000494:pharmacology; D014101:Torpedo; D014403:Tubocurarine / Q000494:pharmacology
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D009498:Neurotoxins; D011950:Receptors, Cholinergic; D030162:Reptilian Proteins; D012910:Snake Venoms; D013388:Succinimides; C045295:methyl alpha-neurotoxin; C011240:dithiobis(succinimidylpropionate); D005033:Ethylmaleimide; D004229:Dithiothreitol; D014403:Tubocurarine