We report the cloning and characterization of rat ␣10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the ␣10 nAChR subunit gene is most similar to the rat ␣9 nAChR, and both ␣9 and ␣10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with ␣10 cRNA alone or in pairwise combinations with either ␣2-␣6 or ␤2-␤4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of ␣9 and ␣10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric ␣9 channels, the ␣9␣10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct currentvoltage relationship, and a biphasic response to changes in extracellular Ca 2؉ ions. The pharmacological profiles of homomeric ␣9 and heteromeric ␣9␣10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both ␣9 and ␣10 subunits.

In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both ␣9 and ␣10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88 -100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16 -P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10 -P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that ␣9 mRNA is expressed in adult IHCs but that ␣10 mRNA disappears after the onset of hearing. In the present study, antibodies to ␣10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of ␣10, but not ␣9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.

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