Mapping the agonist binding site of the nicotinic acetylcholine receptor - Orientation requirements for activation by covalent agonist

To characterize the structural requirements for ligand orientation compatible with activation of the Torpedo nicotinic acetylcholine receptor (nAChR), we used Cys mutagenesis in conjunction with sulfhydryl-reactive reagents to tether primary or quaternary amines at defined positions within the agonist binding site of nAChRs containing mutant alpha- or gamma-subunits expressed in Xenopus oocytes, 4-(N-Maleimido)benzyltrimethylammonium and 2-aminoethylmethanethiosulfonate acted as irreversible antagonists when tethered at alpha Y93C, alpha Y198C, or gamma E57C, as well as at alpha N94C (2-aminoethylmethanethiosulfonate only), [2-(Trimethylammonium)ethyl]-methanethiosulfonate (MTSET), which attaches thiocholine to binding site Cys, also acted as an irreversible antagonist when tethered at alpha Y93C, alpha N94C, or gamma E57C. However, MTSET modification of alpha Y198C resulted in prolonged activation of the nAChR not reversible by washing but inhibitable by subsequent exposure to non competitive antagonists. Modification of alpha Y198C (or any of the other positions tested) by [(trimethylammonium)methyl]methanethiosulfonate resulted only in irreversible inhibition, while modification of alpha Y198C by [3-(trimethylammonium)propyl]methanethiosulfonate resulted in irreversible activation of nAChR, but at lower efficacy than by MTSET, Thus changing the length of the tethering arm by less than 1 Angstrom in either direction markedly effects the ability of the covalent trimethylammonium to activate the nAChR, and agonist activation depends on a very selective orientation of the quaternary ammonium within the agonist binding site.