CONICET | Buscador de Institutos y Recursos Humanos

Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop family of neurotransmitter-gated ion channels. Receptors in the family are formed by five homologous subunits oriented around a central ion channel similar to barrel staves. In vertebrates, nine non muscle a-subunits (a2-a10) and three b-subunits (b2-b4) have been cloned. The reported combinations of subunits that form functional vertebrate nAChRs is expanding and the number of potential molecular forms of nicotinic receptors is very large. Determining the stoichiometry of each association has become challenging in most cases. The a9 and a10 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 nAChR subunits that have been cloned and their identification has established a distant early divergent branch within the nAChR gene family. The a10 subunit serves as a structural component leading to heteromeric a9a10 nAChRs with distinct properties. We have now probed the stoichiometry of recombinant a9a10 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 a9 or a10 subunit bearing a reporter mutation of a valine to threonine at position 13 of the second transmembrane domain. Since the mutation increased the sensitivity of the receptor for acetylcholine (ACh), but mutations at different subunits were not equivalent, the number of a9 and a10 subunits could be inferred from the number of components in compound concentration-response curves to ACh. The results were confirmed through the analysis of the effects of a mutation to threonine at position 17 of TM2. We conclude that the recombinant a9a10 receptor is a pentamer with a (a9)2(a10)3 stoichiometry.