The ␣9 and ␣10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of ␣9 and ␣10 overlaps with that of ␣7 in the cochlea and in sites such as dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes, and skin. We now report the cloning, total synthesis, and characterization of a novel toxin ␣-conotoxin PeIA that discriminates between ␣9␣10 and ␣7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. ␣-Conotoxin PeIA displayed a 260-fold higher selectivity for ␣-bungarotoxin-sensitive ␣9␣10 nAChRs compared with ␣-bungarotoxin-sensitive ␣7 receptors. The IC 50 of the toxin was 6.9 ؎ 0.5 nM and 4.4 ؎ 0.5 nM for recombinant ␣9␣10 and wild-type hair cell nAChRs, respectively. ␣-Conotoxin PeIA bears high resemblance to ␣-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither ␣-conotoxin MII nor ␣-conotoxin GIC at concentrations of 10 M blocked acetylcholine responses elicited in Xenopus oocytes injected with the ␣9 and ␣10 subunits. Among neuronal non-␣-bungarotoxin-sensitive receptors, ␣-conotoxin PeIA was also active at ␣3␤2 receptors and chimeric ␣6/␣3␤2␤3 receptors. ␣-Conotoxin PeIA represents a novel probe to differentiate responses mediated either through ␣9␣10 or ␣7 nAChRs in those tissues where both receptors are expressed.

The ␣9 and ␣10 nicotinic cholinergic subunits assemble to form the receptor that mediates synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea. They are the latest vertebrate nicotinic cholinergic receptor (nAChR) subunits that have been cloned, and their identification has established a distant early divergent branch within the nAChR gene family. The ␣10 subunit serves as a "structural" component leading to heteromeric ␣9␣10 nAChRs with distinct properties. We now have probed the stoichiometry of recombinant ␣9␣10 nAChRs expressed in Xenopus oocytes. We have made use of the analysis of the population of receptors assembled from a wild-type subunit and its partner ␣9 or ␣10 subunit bearing a reporter mutation of a valine to threonine at position 13Ј of the second transmembrane domain (TM2). Because the mutation increased the sensitivity of the receptor for acetylcholine (ACh) but mutations at different subunits were not equivalent, the number of ␣9 and ␣10 subunits could be inferred from the number of components in compound concentration-response curves to ACh. The results were confirmed via the analysis of the effects of a mutation to threonine at position 17Ј of TM2. Because at this position the mutations at different subunits were equivalent, the stoichiometry was inferred directly from the shifts in the ACh EC 50 values. We conclude that the recombinant ␣9␣10 receptor is a pentamer with a (␣9) 2 (␣10) 3 stoichiometry.

The expansion and pruning of ion channel families has played a crucial role in the evolution of nervous systems. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels with distinct roles in synaptic transmission at the neuromuscular junction, the central and peripheral nervous system, and the inner ear. Remarkably, the complement of nAChR subunits has been highly conserved along vertebrate phylogeny. To ask whether the different subtypes of receptors underwent different evolutionary trajectories, we performed a comprehensive analysis of vertebrate nAChRs coding sequences, mouse single-cell expression patterns, and comparative functional properties of receptors from three representative tetrapod species. We found significant differences between hair cell and neuronal receptors that were most likely shaped by the differences in coexpression patterns and coassembly rules of component subunits. Thus, neuronal nAChRs showed high degree of coding sequence conservation, coupled to greater coexpression variance and conservation of functional properties across tetrapod clades. In contrast, hair cell α9α10 nAChRs exhibited greater sequence divergence, narrow coexpression pattern, and great variability of functional properties across species. These results point to differential substrates for random change within the family of gene paralogs that relate to the segregated roles of nAChRs in synaptic transmission.

Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.