Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism

The catecholamine release-inhibitory chromogranin A fragment catestatin (ch romogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cho linergic signaling in chromaffin cells. A previous homology model of catest atin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we foun d that [I-125]-catestatin binds to the surface of intact PC12 and bovine ch romaffin cells with high affinity (K-D = 15.2+/-1.53 nM) and specificity (l ack of displacement by another [N-terminal] fragment of chromogranin A). Ni cotinic agonist (carbamylcholine) did not displace [I-125]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [H-3]-e pibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consis tent with catestatin's non-competitive nicotinic mechanism. [I-125]-catesta tin could be displaced from chromaffin cells by substance P (IC50 similar t o5 muM), though at far lower potency than displacement by catestatin itself (IC50 similar to 350-380 nM), suggesting that catestatin and substance P o ccupy an identical or overlapping non-competitive site on the nicotinic rec eptor, at different affinities (catestatin > substance P). Small, non-pepti de non-competitive nicotinic antagonists (hexamethonium or clonidine) did n ot diminish [I-125]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic S-125]-catestatin were observed on chromaff in receptor fur peptide and non-peptide antagonists. Similar binding and in hibitory profiles for [ cells as well as nicotinic receptor-enriched Torped o membranes. Covalent cross-linking of [I-125]-catestatin to Torpedo membra nes suggested specific contacts of [I-125]-catestatin with the delta, gamma , and beta subunits of the nicotinic receptor, a finding consistent with pr ior homology modeling of the interaction of catestatin with the extracellul ar face of the nicotinic heteropentamer. We conclude that catestatin occlud es the nicotinic cation pore by interacting with multiple nicotinic subunit s at the pore vestibule. Such binding provides a physical explanation for n on-competitive antagonism of the peptide at the nicotinic receptor. (C) 200 0 Elsevier Science B.V. All rights reserved.