Identification of the subunits of the nicotinic cholinergic receptors in the rat cochlea using RT–PCR and in situ hybridization (original) (raw)
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Proceedings of the National Academy of Sciences, 2001
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. ¶ To whom reprint requests should be addressed at
Proceedings of the National Academy of Sciences, 2001
We report the cloning and characterization of rat α10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the α10 nAChR subunit gene is most similar to the rat α9 nAChR, and both α9 and α10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with α10 cRNA alone or in pairwise combinations with either α2-α6 or β2-β4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of α9 and α10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric α9 channels, the α9α10 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 α9 and heteromeric α9α10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous choli...
Developmental mRNA expression of the α10 nicotinic acetylcholine receptor subunit in the rat cochlea
Developmental Brain Research, 2002
A recently discovered a10 subunit of the nicotinic acetylcholine receptor (nAChR) family is believed to form a heteromeric receptor with the a9 nAChR subunit in auditory hair cells. In the present study, the a10 nAChR subunit expression in the developing and adult rat inner ear was analyzed by PCR and localized using isotopic in situ hybridization. Unlike the a9 subunit, the a10 subunit was not detected at embryonic day 18 (E18). From E21 through postnatal day 15 (P15), the a10 subunit was localized over both inner hair cell (IHC) and outer hair cell (OHC) regions, but in the mature cochlea detectable levels of a10 mRNA were found only over the OHC region. From E21 through adult ages, there was also a small but consistent basal to apical gradient of a10 expression; that is, higher levels in basal regions and lower levels in apical regions. Previously, we detected the a9 nAChR subunit over IHCs as early as E18 and throughout adult ages with a clear basal-apical gradient of expression. Our studies raise the question of whether the a9 and a10 subunits are differentially regulated during embryonic and postnatal development.
Molecular Brain Research, 1998
The expression of the a 9 nicotinic acetylcholine receptor nAChR subunit was investigated in perinatal and adult rat cochleae using w 35 x S labeled cRNA in situ hybridization techniques. In the adult, a 9 expression showed both longitudinal and radial gradients. The Ž. highest expression occurs over outer hair cells OHCs in basal regions, and particularly, OHCs in row 1. In contrast, expression over IHCs is lowest in basal regions and highest in apical regions. During embryonic and postnatal ages, the pattern of a 9 expression differs. Expression of a 9 was nearly equivalent over IHCs and OHCs. Additionally, the greater epithelial ridge, which is adjacent to IHCs before birth, shows a high level of a 9 expression. These data are consistent with current models of efferent synaptogenesis and suggest that the expression of the a 9 nAChR may be influenced by the arrival of efferent axons. q 1998 Elsevier Science B.V.
The Journal of physiology, 2005
Before the onset of hearing, a transient efferent innervation is found on inner hair cells (IHCs). This synapse is inhibitory and mediated by a nicotinic cholinergic receptor (nAChR) probably formed by the alpha9 and alpha10 subunits. We analysed the pharmacological and biophysical characteristics of the native nAChR using whole-cell recordings from IHCs in acutely excised apical turns of the rat organ of Corti. Nicotine did not activate but rather blocked the acetylcholine (ACh)-evoked currents with an IC50 of 1 +/- 0.1 microM. Antagonists of non-cholinergic receptors such as strychnine, bicuculline and ICS-205930 blocked ACh-evoked responses with an IC50 of 8.6 +/- 0.8 nM, 59 +/- 4 nM and 0.30 +/- 0.02 microM, respectively. The IHC nAChR was both permeable to (P(Ca)/P(Na) = 8 +/- 0.9) and modulated by external Ca2+. ACh-evoked currents were potentiated by Ca2+ up to 500 microM but were reduced by higher concentrations of this cation. Ba2+ mimicked the effects of Ca2+ whereas Mg2+ ...
Proceedings of the National Academy of Sciences, 2007
Although homomeric channels assembled from the ␣9 nicotinic acetylcholine receptor (nAChR) subunit are functional in vitro, electrophysiological, anatomical, and molecular data suggest that native cholinergic olivocochlear function is mediated via heteromeric nAChRs composed of both ␣9 and ␣10 subunits. To gain insight into ␣10 subunit function in vivo, we examined olivocochlear innervation and function in ␣10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8 -9 inner hair cells revealed ACh-gated currents in ␣10 ؉/؉ and ␣10 ؉/؊ mice, with no detectable responses to ACh in ␣10 ؊/؊ mice. In contrast, a proportion of ␣10 ؊/؊ outer hair cells showed small ACh-evoked currents. In ␣10 ؊/؊ mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual ␣9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, ␣10 ؊/؊ mice exhibit both an abnormal olivocochlear morphology and innervation to outer hair cells and a highly disorganized efferent innervation to the inner hair cell region. Our results demonstrate that ␣9 ؊/؊ and ␣10 ؊/؊ mice have overlapping but nonidentical phenotypes. Moreover, ␣10 nAChR subunits are required for normal olivocochlear activity because ␣9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in ␣10 ؊/؊ mutant mice.
Proceedings of the National Academy of Sciences of the United States of America, 2001
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 ind...
Immunolocalization of α4 and α7 subunits of nicotinic receptor in rat cochlear nucleus
Hearing Research, 1999
The rat cochlear nucleus (CN) is known to receive cholinergic input. To investigate the prevalence of nicotinic acetylcholine receptor (nAChR), immunohistochemistry for K K4 and K K7 subunits, which represent nAChRs with high binding affinities for nicotine and K K-bungarotoxin, respectively, was performed on perfusion-fixed rat brain sections. Microscopic observations and densitometric measurements show dense labeling for K K7 but not K K4. Within the CN, K K7 receptors are found in all subregions, with relatively high densities in granular regions. The distribution of K K7 within the CN appears to correlate more closely with that of acetylcholinesterase than with mAChR or choline acetyltransferase. Our results suggest a role of nicotinic cholinergic transmission in the rat CN associated with high affinity for K K-bungarotoxin.
Journal of the Association for Research in Otolaryngology, 2009
Efferent inhibition of cochlear hair cells is mediated by α9α10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express α10 into adulthood by expressing the α10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the α10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 α10 transgenic mice backcrossed to a Chrna10 −/− background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the α10 transgene restored nAChR function. However, in the α10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental downregulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.
Cell, 1994
We report the isolation and functional characterization of a member of the nicotinic acetylcholine receptor subunit gene family, cc9. Xenopus oocytes injected with a9 cRNA express a homomeric receptor-channel complex that is activated by acetylcholine. The a9 receptor displays an unusual mixed nicotinic-muscarinic pharmacological profile. The unique properties of the a9 receptor-channel complex closely match those described for the cholinergic receptor present in vertebrate cochlear hair cells. In situ hybridization studies reveal a restricted pattern of a9 gene expression that includes the outer hair cells of the rat cochlea. Our results suggest that the cc9 receptor is involved in the cholinergic efferent innervation of cochlear hair cells and thus may modulate the encoding of auditory stimuli.