Expression of the nicotinic acetylcholine receptor subunit, alpha9, in the guinea pig cochlea.

Abstract

Acetylcholine is a major neurotransmitter of the cochlear efferent system. Based on its high level of expression in hair cells, the recently cloned nicotinic receptor subunit, alpha9 [Elgoyhen et al., Cell 79 (1994) 705-715], is likely to be the postsynaptic receptor for acetylcholine in hair cells either as a homomeric complex or with other subunits yet to be identified. To further study this receptor, we cloned and sequenced alpha9 cDNA from the guinea pig organ of Corti library [Wilcox and Fex, Hear. Res. 62 (1992) 124-126]. The sequence of the guinea pig alpha9 cDNA is similar to that of the rat, with identities of 85% and 89% at the nucleotide and amino acid levels, respectively. Most differences are in the cytoplasmic loop domain between the transmembrane segments 3 and 4. We also observed minor differences in the putative ligand binding regions. Pharmacological differences between acetylcholine receptors on outer hair cells of rat and guinea pig have been reported, and the minor structural changes we observe could account for these differences. Reverse transcription-polymerase chain reaction analysis showed a high expression of alpha9 in the organ of Corti while expression was low or not detected in the spiral ganglion. In situ hybridization histochemistry showed expression of alpha9 mRNA in both inner and outer hair cells, with much higher expression in outer hair cells than in inner hair cells. In the inner hair cell, silver grains were more abundant over the basal part of the cell than over the apical part. Immunocytochemistry showed a pattern of distribution of the alpha9 protein similar to that seen for mRNA with in situ hybridization. Immunolabeling was most intense at the bases of both inner and outer hair cells. To determine the effect of hair cell loss on alpha9 expression, hair cells were destroyed by either systemic or local application of kanamycin. This treatment led to a down regulation of alpha9 in hair cells; this down regulation appeared to precede hair cell degeneration. In the spiral ganglion, a transient up regulation of alpha9, as determined by RT-PCR, was seen 4-6 weeks after kanamycin treatment.