alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells

Abstract

We report the cloning and characterization of rat alpha10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the alpha10 nAChR subunit gene is most similar to the rat alpha9 nAChR, and both alpha9 and alpha10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with alpha10 cRNA alone or in pairwise combinations with either alpha2-alpha6 or beta2-beta4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of alpha9 and alpha10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric alpha9 channels, the alpha9alpha10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca(2+) ions. The pharmacological profiles of homomeric alpha9 and heteromeric alpha9alpha10 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 alpha9 and alpha10 subunits.

Figure 1

Nucleotide and deduced amino acid sequences of a cDNA clone encoding the rat nAChR α10 subunit. The deduced amino acid sequence is shown above the nucleotide sequence. Amino acids are presented as the standard single-letter code. Functional domains are underlined and include a possible signal peptide and four putative transmembrane regions. Filled circles (●) are placed above extracellular cysteine residues that are conserved in nAChR agonist-binding (or α-type) subunits. The calculated molecular mass of the mature nonglycosylated form of the rat α10 protein is 47,127 Da. Intron positions are indicated by an arrowhead, and the asterisk marks the translation termination codon.

Figure 2

α9α10 nAChRs exhibit distinct functional properties. (A, Left) Representative electrophysiological responses to 100 μM ACh obtained in an oocyte expressing the α9α10 nAChR before (Upper) and after (Lower) a 4-h incubation in 100 μM BAPTA-AM. (Right) Voltage ramps (2 sec from −120 to +50 mV, at V_hold = −70 mV) were applied 20 sec after the onset of 100 μM ACh application. (B) Representative responses elicited by ACh (V_hold = −90 mV) in the presence of different concentrations of extracellular Ca²⁺ for oocytes expressing α9α10 nAChRs (Upper) and α9 nAChRs (Lower). (C) Responses to ACh either in the continued presence of agonist or after repeated challenges with agonist (10-sec pulses with 40-sec intervals). (Upper) α9α10 nAChRs. (Lower) α9 nAChRs.

Figure 3

Pharmacological responses of oocytes expressing the α9α10 nAChR. (Left) Concentration-response curves for ACh 1, 1-dimethyl-4-phenylpiperazinium (DMPP) and oxotremorine methiodide (Oxo-M). (Middle and Right) Inhibition of ACh-evoked responses in the presence of a variety of cholinergic and noncholinergic receptor compounds. The estimated IC₅₀ values for each drug are summarized in Table 1.

Figure 4

α10 nAChR subunit gene expression in cochlear and vestibular hair cells. (A) At embryonic day 20, both inner and outer hair cells (arrows) were decorated with silver grains after emulsion dipping of slides. No other structures in the cochlea were labeled (magnification ×630). (B) In adults (2–4 months old), α10 mRNA was detected only in outer hair cells and not in inner hair cells. BM, basilar membrane; IHC, inner hair cells; OHC, outer hair cells (magnification ×1,000). (C) Adult cristae express α10 transcripts as evidenced by the dense accumulation of silver grains over the entire surface (see asterisks) of the sensory end organ. Note the lack of positive reaction at both the transitional epithelium (TE) and the supporting cell layer (black arrows) underlying the hair cell layer (magnification ×630).