Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to alpha-bungarotoxin

We have investigated the molecular determinants responsible for cy-bungarot oxin (alpha Bgtx) binding to nicotinic acetylcholine receptors through chim eric analysis of two homologous alpha subunits, one highly sensitive to alp ha Bgtx block (alpha 1) and the other, alpha Bgtx-insensitive (alpha 3). By replacing rat (alpha 3) residues 184-191 with the corresponding region fro m the Torpedo alpha 1 subunit, we introduced a cluster of five alpha 1 resi dues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-lgl) into the (alpha 3 su bunit. Functional activity and alpha Bgtx sensitivity were assessed followi ng co-expression in Xenopus oocytes of the chimeric alpha 3 subunit (alpha 3/alpha 1[5]) with either rat beta 2 or beta 4 subunits. Agonist-evoked res ponses of alpha 3/alpha 1[5]-containing receptors were blocked by alpha Bgt x with nanomolar affinity IC50 values: 41 nM for (alpha 3/alpha 1[5]beta 2 and 19 nM for alpha 3/alpha 1[5]beta 4). Furthermore, receptors containing the single point mutation alpha 3K189Y acquire significant sensitivity to a lpha Bgtx block ICS, values: 186 nM for alpha 3K189Y beta 2 and 179 nM for (alpha 3K189Y beta 4), Another alpha 3 chimeric subunit, alpha 3/alpha 7[6] , similar to alpha 3/alpha 1[5] but incorporating the corresponding residue s from the alpha Bgtx-sensitive cur subunit, also conferred potent alpha Bg tx sensitivity to chimeric receptors when co-expressed with the beta 4 subu nit (IC50 value = 31 nM). Our findings demonstrate that the residues betwee n positions 184 and 191 of the alpha Bgtx-sensitive subunits al and alpha 7 play a critical functional role in the interaction of alpha Bgtx with nico tinic acetylcholine receptors sensitive to this toxin.