Constitutive Expression of the α10 Nicotinic Acetylcholine Receptor Subunit Fails to Maintain Cholinergic Responses in Inner Hair Cells After the Onset of Hearing

Abstract

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.

Generation and Characterization of α9 and α10 Nicotinic Acetylcholine Receptor Subunit Knockout Mice on a C57BL/6J Background

“…The results are somewhat at odds with previous data obtained using a α9 KO generated by the deletion of exon 4 (and its flanking intronic sequences) and backcrossed onto a mixed CBA/CaJ X 129Sv background. In initial studies, it was concluded that the deletion of α9 was sufficient to render the α9α10 nAChR incapable of carrying current (Vetter et al, 2007 ; Taranda et al, 2009 ). Subsequent studies comparing α9 KOs with WTs (e.g., Mohammadi and Christie, 2014 ; Terreros et al, 2016 ; Mohammadi et al, 2017 ; Tu et al, 2017 ), have typically not used α10 KO mice, but presumed that deletion of α9 is equivalent to a deletion of the α9α10 receptor.…”

“…The contribution of mammalian α9 and α10 to complementary binding sites is nonequivalent (Boffi et al, 2017 ), thus rendering the mammalian α10 incapable of forming a homomer that conducts current (Franchini and Elgoyhen, 2006 ; Lipovsek et al, 2012 ). In a recent study, it was demonstrated, however, that both α9 and α10 subunits contribute to the principal component of the agonist binding site, contradicting the hypothesis that the α10 is a structural subunit, but confirming that the integrity of both subunits is necessary for the wildtype receptor function in mammals (Sgard et al, 2002 ; Vetter et al, 2007 ; Taranda et al, 2009 ; Boffi et al, 2017 ).…”

Cochlear efferent innervation and function

“…α10ACh receptors on IHCs normally disappear with the disappearance of MOC innervation. Genetically-manipulated mice that have α10AChRs in adulthood do not show ACh currents, presumably because IHC SK2 channels are also down-regulated [70]. Experiments in α10AChR-knockout animals show that the development of basic IHC properties does not require the α10AChR subunit [71].…”

The efferent medial olivocochlear-hair cell synapse

“…The fact that cholinergic responses are completely absent in hair cells from these SK2 null mice, even though the amount of α9 and α10 mRNA, as evaluated by quantitative RT-PCR does not differ from those in wild-type animals (Kong et al, 2008; Murthy et al, 2009a), strongly suggests that SK2 is fundamentally required for the assembly, trafficking, and/or anchorage of the nAChR macromolecular synaptic complex to the membrane. This is further supported by results derived from the analysis of a line of mice that constitutively express the α10 subunit (Taranda et al, 2009a). Using this same SK2 knockout animal model, SK2 channels have been shown to be necessary for the long-term survival of MOC fibers and synapses (Murthy et al, 2009a).…”