title: 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.
abstract: The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.
authors.affiliation: Department of Medicine and Center for Molecular Genetics, University of California, and Veteran Affairs San Diego Healthcare System, 92161-9111H, San Diego, CA, USA. ltaupenot@ucsd.edu
authors.forename: L
authors.initials: L
authors.lastname: Taupenot
authors.forename: S K
authors.initials: SK
authors.lastname: Mahata
authors.forename: M
authors.initials: M
authors.lastname: Mahata
authors.forename: R J
authors.initials: RJ
authors.lastname: Parmer
authors.forename: D T
authors.initials: DT
authors.lastname: O'Connor
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000493:pharmacokinetics / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D019439:Chromaffin Cells / Q000187:drug effects / Q000502:physiology*; D053379:Chromogranin A; D002864:Chromogranins / Q000493:pharmacokinetics / Q000494:pharmacology*; D004557:Electric Organ / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D018722:Nicotinic Agonists / Q000493:pharmacokinetics*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000493:pharmacokinetics / Q000494:pharmacology*; D011725:Pyridines / Q000493:pharmacokinetics / Q000494:pharmacology; D051381:Rats; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000502:physiology*; D014101:Torpedo
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D053379:Chromogranin A; D002864:Chromogranins; D018722:Nicotinic Agonists; D010446:Peptide Fragments; D011725:Pyridines; D011978:Receptors, Nicotinic; C109084:chromogranin A (344-364); C082748:epibatidine