IDENTIFICATION AND FUNCTIONAL-CHARACTERIZATION OF A NEW AGONIST SITE ON NICOTINIC ACETYLCHOLINE-RECEPTORS OF CULTURED HIPPOCAMPAL-NEURONS

Electrophysiological and biochemical techniques were used to demonstra te that alpha-bungarotoxin-, methyllycaconitine-sensitive neuronal nic otinic acetylcholine receptors (nAChRs) can be activated via a novel a gonist site(s). The residue proposed to be essential to this site is t he amino acid Lys-1 25 of the receptor alpha subunit. In outside-out p atches excised from cultured hippocampal neurons, physostigmine (PHY) and 1-methyl-PHY activated single channels whose main conductances wer e 46 and 23 pS. This action was insensitive to DL-2-amino-5-phosphonov aleric acid, atropine, tetrodotoxin and competitive nicotinic antagoni sts, but blocked by benzoquinonium or FK1, a nAChR-specific antibody r aised against rat muscle nAChR alpha subunits that binds to the novel site. Indirect immunofluorescence staining demonstrated that FK1 binds to the hippocampal neurons, as would be expected based on the high de gree of homology among nAChR alpha subunits from diverse sources in th e region surrounding Lys-125. PHY prevented the binding of FK1, thus s upporting that FK1 is a specific probe for the PHY site. High-affinity sites (K(D) almost-equal-to 35 nM) for 1-methyl-PHY were identified i n hippocampal neurons Similar to PHY, benzoquinonium (0.1-10 muM) and galanthamine (1-10 muM) activated nicotinic single channels. The agoni sts benzoquinonium and PHY were also open-channel blockers at the neur onal nAChRs, whereas galanthamine was predominantly a desensitizing ag ent. In mouse fibroblasts transfected with cDNAs of alpha4 and beta2 n euronal nAChR subunits, PHY also activated single channels that were b locked by FK1. In these cells, dihydro-beta-erythroidine blocked singl e channels activated by (+)-anatoxin-a and did not affect those opened by PHY. Thus, the present results suggest that the novel agonist site located on the receptor alpha subunit is a common feature of neuronal nAChRs.