Abstract

The binding of [3H]substance P to nicotinic acetylcholine receptor-enriched Torpedo electroplaque membranes was characterized. In the absence of cholinergic agonist, [3H]substance P binding was displaced by unlabeled substance P with an IC50 of 31 +/- 7 microM. In the presence of 10 mM carbamylcholine, displacement reflected two populations of binding sites with IC50 values of 0.93 +/- 0.39 and 30 +/- 5 microM, with the higher affinity component contributing 69 +/- 2% of the inhibition. Equilibrium binding parameters were calculated by transformation of the concentration dependences of inhibition into saturation isotherms. In the absence of agonist, substance P bound with a Kd of 42 +/- 7 microM to 3-4 sites/alpha-bungarotoxin binding site. In the presence of agonist, substance P bound to two sites, a low affinity site not significantly different from that seen in the absence of agonist (Kd = 25 +/- 8 microM, approximately 3 sites/alpha-bungarotoxin site) and a high affinity site with a Kd of 0.55 +/- 0.32 microM (approximately 1 site/2 alpha-bungarotoxin sites, 1 site/receptor). The increase in substance P binding induced by carbamylcholine was blocked by the nicotinic antagonists alpha-bungarotoxin and d-tubocurarine but was not affected by the muscarinic antagonist atropine. The concentration dependence of the carbamylcholine-induced increase had two components, with EC50 values for the agonist of 9.1 +/- 4.2 microM (56 +/- 16% of the increase) and 1.3 +/- 0.5 mM. The structural specificity of agonist-dependent high affinity substance P binding was identical to that seen for inhibition of nicotinic receptor activation and substantially different from that of binding to the G protein-coupled tachykinin receptors. From the time courses of association, it appears that substance P binds preferentially to a transient agonist-induced receptor state. The gamma and delta subunits of the receptor were specifically labeled in an agonist-dependent manner after cross-linking of [3H]substance P to the receptor with the bifunctional cross-linking reagent bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone or after photoaffinity labeling of the receptor with 125I-p-benzoylphenylalanine-substance P. These results demonstrate the existence of a high affinity agonist-induced binding site for substance P on the nicotinic acetylcholine receptor that probably mediates the noncompetitive inhibition by the peptide of receptor activation.