Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies

Abstract

Neuronal acetylcholine receptors (AChRs), which bind nicotine with high affinity but do not bind α-bungarotoxin (αBgt), have recently been immunoaffinity-purified from chicken (Whiting and Lindstrom, 1986a) and rat (Whiting and Lindstrom, 1987a) brain using monoclonal antibodies (mAbs). Here we report the characterization of nicotinic AChRs of bovine and human brain using as probes mAbs prepared to AChRs from rat brain. Both the human and bovine brain AChRs exhibit high affinity for L-nicotine (K(i) = 16 nM for bovine AChR and K(i) = 6.5 nM for human AChR) and other cholinergic agonists, relatively lower affinity for cholinergic antagonists, and do not bind αBgt. These AChRs are affinity-labeled with bromoacetylcholine and 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA) after reduction with dithiothreitol, indicating that amino acid residues homologous to cysteines 192 and 193 of α subunits of Torpedo electric organ AChRs are conserved. Immunoaffinity-purified bovine brain AChR consists of 2 types of subunit, M(r) 50,600 and M(r) 74,400. The M(r) 74,400 subunit was affinity-labeled with 3H-MBTA, indicating that it contains the ACh binding site. Thus, mAbs have proven to be excellent probes for these proteins, and have been used to show that neuronal nicotinic AChRs of chickens, rats, and cattle are macromolecules approximately 10 S in size and composed of only 2 kinds of subunits; an ACh-binding subunit and a structural subunit.