Indurthi_2014_Biochem.Pharmacol_89_131

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in fast synaptic transmission. nAChRs are pentameric receptors formed from a combination of different or similar subunits to produce heteromeric or homomeric channels. The heteromeric, alpha9alpha10 nAChR subtype is well-known for its role in the auditory system, being expressed in cochlear hair cells. These nAChRs have also been shown to be involved in immune-modulation. Antagonists of alpha9alpha10 nAChRs, like the alpha-conotoxin Vc1.1, have analgesic effects in neuropathic pain. Unlike other nAChR subtypes there is no evidence that functional receptor stoichiometries of alpha9alpha10 exist. By using 2-electrode voltage clamp methods and maintaining a constant intracellular Ca(2+) concentration, we observed a biphasic activation curve for ACh that is dependent on receptor stoichiometry. Vc1.1, but not the alpha9alpha10 antagonists RgIA or atropine, inhibits ACh-evoked currents in a biphasic manner. Characteristics of the ACh and Vc1.1 activation and inhibition curves can be altered by varying the ratio of alpha9 and alpha10 mRNA injected into oocytes, changing the curves from biphasic to monophasic when an excess of alpha10 mRNA is used. These results highlight the difference in the pharmacological profiles of at least two different alpha9alpha10 nAChR stoichiometries, possibly (alpha9)(3)(alpha10)(2) and (alpha9)(2)(alpha10)(3). As a result, we infer that there is an additional binding site for ACh and Vc1.1 at the alpha9-alpha9 interface on the hypothesized (alpha9)(3)(alpha10)(2) nAChR, in addition to the alpha10-alpha9 and or alpha9-alpha10 interfaces that are common to both stoichiometries. This study provides further evidence that receptor stoichiometry contributes another layer of complexity in understanding Cys-loop receptors.