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

Abstract

We have investigated the molecular determinants responsible for α- bungarotoxin (αBgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous α subunits, one highly sensitive to αBgtx block (α1) and the other, αBgtx-insensitive (α3). By replacing rat α3 residues 184-191 with the corresponding region from the Torpedo α1 subunit, we introduced a cluster of five α1 residues (Trp-184, Trp-187, Val- 188, Tyr-189, and Thr-191) into the α3 subunit. Functional activity and αBgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric α3 subunit (α3/α1[5]) with either rat β2 or β4 subunits. Agonist-evoked responses of α3/α1[5]-containing receptors were blocked by αBgtx with nanomolar affinity (IC50 values: 41 nM for α3/α[5]β2 and 19 nM for α3/α1[5]β4). Furthermore, receptors containing the single point mutation α3K189Y acquire significant sensitivity to αBgtx block (IC50 values: 186 nM for α3K189Yβ2 and 179 nM for α3K189Yβ4). Another α3 chimeric subunit, α3/α7[6], similar to α3/α1[5] but incorporating the corresponding residues from the αBgtx-sensitive α7 subunit, also conferred potent αBgtx sensitivity to chimeric receptors when co-expressed with the β4 subunit (IC50 value = 31 nM). Our findings demonstrate that the residues between positions 184 and 191 of the αBgtx-sensitive subunits α1 and α7 play a critical functional role in the interaction of αBgtx with nicotinic acetylcholine receptors sensitive to this toxin.