Α9α10 Acetylcholine Receptors: Structure and Functions

Neurotransmitter 2016; 3: e1298. doi: 10.14800/nt.1298; © 2016 by Ana E. Vázquez http://www.smartscitech.com/index.php/nt Special Issue Edited by Dr. Hugo R. Arias| Functional and therapeutic aspects of the nicotinic acetylcholine receptor family.

REVIEW

α9α10 acetylcholine receptors: structure and functions

Ana E. Vázquez

Previously at the Research Laboratories Department of Otolaryngology- Head and Neck Surgery, University of California, Davis, CA 95616, USA

Correspondence: Ana E. Vázquez E-mail: [email protected] Received: April 03, 2016 Published online: May 12, 2016

The α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits assemble as pentameric complexes with a central ligand-gated cation-selective pore. However, these subunits do not assemble with other nAChR subunits. The general topology of each subunit is similar to that of other ligand-gated, Cys-loop ion channels. Although the sequence and genomic organization of the α9 and α10 subunits determined their classification as nicotinic subunits, α9α10 nAChRs exhibit peculiar pharmacological properties that are not common to other nicotinic or muscarinic receptors. α9α10 nAChRs are expressed in diverse tissues, including auditory sensory hair cells, afferent neurons in the vestibular sensory organs, neurons of the dorsal root ganglion, skin, and bronchial epithelia. Furthermore, it has become apparent that nAChRs containing α9 and α10 subunits play essential roles in various physiological processes. For example, α9 is required for the normal development of the efferent synapses in the inner ear, and after the onset of hearing α9α10 nAChRs serve as the unique source of extracellular calcium for Ca2+-activated potassium channels in mature auditory hair cells. The development of drugs that selectively target α9/α10 receptors promises to be of therapeutic benefit since these receptors have distinct physiologic functions. They hold promise as pharmacological targets to treat complex auditory and vestibular pathologies, neuropathic pain, and pathologies of the skin and bronchia. Keywords: α9α10 nicotinic acetylcholine receptors; cochlea; efferent; hair cell; nicotinic; vestibular To cite this article: Ana E. Vázquez. α9α10 acetylcholine receptors: structure and functions. Neurotransmitter 2016; 3: e1298. doi: 10.14800/nt.1298.

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Introduction nAChRs were initially thought to be expressed exclusively in neurons and neuronal tissues, however, it has recently The nicotinic acetylcholine receptors (nAChRs) are become clear that many of them are also expressed and play members of the superfamily of ligand-gated, Cys-loop ion critical roles in other tissues. channels. Like the muscle nAChR, prototype of this superfamily, neuronal nAChRs consist of a complex of five This review focuses on recent findings related to the homologous subunits lining a central cation-permeable pore. structure and function of α9α10 nAChRs. α9 and α10 are the Neuronal nAChRs are pentameric receptors formed by only most recently cloned members of the nAChR family. The one α subunit type (homopentamers) or by a combination of discoveries that the α9 and shortly after the α10 nAChR different α and β subunits (heteropentamers). Seven α subunit were expressed in sensory hair cells were exciting subunits (α2-α7, α9, and α10) and three β subunits (β2-β4) hallmarks of hearing and vestibular research [3, 4]. have been cloned and studied extensively in mammals [1, 2].

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There are two types of hair cells in the mammalian deduced amino acid sequences for the rat subunits α1-α7 and auditory sensory epithelia. The inner hair cells, arranged in a α9, and the chicken subunit α8 shows that the α9 subunit single row along the cochlea spiral, contact all the afferent shares about 36 to 39% of the amino acid with any of these fibers and are the primary transducers of sound. The other nAChR subunits [4]. However, 57% overall amino acid second type is the outer hair cells, which are arranged as sequence identity is shared between the α9 and α10 subunits, three rows. The outer hair cells are capable of shortening indicating that they are more closely related to each other and lengthening their shape and have been proposed to work than to the muscle nAChRs, to the α7-α8, or to the α2-α6 as small springs modifying the movement of the basilar subfamilies [3, 14]. Additionally, the α9 and α10 subunits have membrane. All hair cells have apical stereocilia that harbors the same genomic organization. For instance, the position of the transduction machinery able to convert mechanical their introns as related to the coding sequence is identical, stimuli into the neural signals which are sent to the brain. but differs to that found for all others nAChRs. In all This transduction machinery is exquisitely sensitive; previously cloned nAChRs, the boundaries of the first four responsive to displacements as small as one nm [reviewed in exons are conserved but in the α9 and α10 subunits, exons III [5]]. However, in addition, auditory perception is directly and IV are continuous [3, 4, 15]. modulated by a sound-evoked feedback mechanism via the olivocochlear efferent system which contributes to frequency Structure of α9α10 nicotinic acetylcholine receptors selectivity and protects the cochlea from damage due to excessive noise. Based on where the neurons are located, nAChRs containing the α9 and α10 subunits are there are lateral and medial components to this efferent assemblies of five subunits, each with the general topological system. The medial olivochoclear system neurons originate organization common to the nAChR family. This in the medial portion of the superior olivary complex and organization includes an extracellular N-terminal domain of project into the cochlea to make synapses at the base of outer about 200 amino acids containing the signature Cys-loop as hair cells [6, 7]. part of the ligand binding site observed in nAChRs [16, 17]. This domain is then followed by three transmembrane A major neurotransmitter at these synapses is domains (TM1-TM3), a cytoplasmic domain, and a fourth acetylcholine (ACh) [reviewed in [8]]. In electrophysiological transmembrane domain (TM4), connecting to the studies of isolated outer hair cells, native nAChRs displayed extracellular carboxyl-terminal domain [17]. intriguing nicotinic but also muscarinic pharmacological properties [9-12]. Following ACh application to isolated outer Intensive research has provided a great deal of information hair cells, outward K+ currents were recorded using the regarding the amino acids and the specific structure of the whole cell voltage and current clamp configurations. These N-terminal extracellular domain which contains the binding ACh-induced currents were demonstrated to be dependent on sites for ACh and other agonists and antagonists in muscle extracellular Ca2+. Under the same conditions, outward and neuronal nAChRs [17-19]. The alignment of the protein currents were also induced by suberyldicholine, carbachol, sequences of the α9 subunit with other previously cloned and dimethylphenylpiperazinium, when these agents were nAChR subunits demonstrated that in this N-terminal tested at high concentrations, however, nicotine, cytisine, and extracellular domain about 25% of the amino acids are muscarine did not produce any effect. The response to ACh conserved [4]. In contrast, when comparing just the α9 and was blocked by low concentrations of classical nicotinic α10 subunits, 67% of the amino acid sequence of this domain antagonists as well as non-classical nicotinic and muscarinic is identical [3, 4]. The significant sequence divergence of this antagonists. For example, strychnine, curare, bicuculline, domain in α9α10 nAChRs with respect to other nAChRs is α-bungarotoxin, and trimethaphan blocked the response at expected to be a major factor underlying the distinct low concentrations, whereas higher concentrations of pharmacological profile of α9α10 nAChRs. However, the atropine, 4-DAMP, AF-DX 116, pirenzepine muscarinic molecular basis for these differences remains to be antagonists were required to block the response. Based on investigated. these pharmacological studies, the receptor expressed in outer hair cells was thought to be nicotinic [11, 13]. This was The trasmembrane domains are the most highly conserved confirmed when the α9 and α10 subunits were cloned and regions of the functional domains in the nAChR. Cysteine their amino acid sequences demonstrated that they belonged substitution followed by accessibility studies indicated early to the Cys-loop ligand-gated ion channel superfamily and not on that the side chains of amino acids in TM2 line the pore to the G-protein coupled receptor superfamily [3, 4]. [20]. Amino acids in TM2 have been demonstrated to play Interestingly, the α9 and α10 subunits seem to define a important roles on gating and on the ion selectivity in other different subfamily of nAChRs. The alignment of the ligand-gated ion channels, including different nAChRs.

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Figure 1. Best alignment of the cytoplasmic domain’s amino acid sequences between TM3 and TM4 of the 7, 9, 10, and 3 subunits. For each nAChR subtype, there is high degree of conservation amongst the species. However, this domain exhibits the most sequence diversity between nAChR subtypes and consists of distinct subdomains (highlighted in gray) highly conserved amongst species. There are five conserved amino acids amongst all subunits (bold white on black background). The alignment was performed using EMBL’s ClustalW2.

Using site-directed mutagenesis, in the same manner that it in the 910 nAChR pentamer [23]. had been demonstrated for the α7 nAChR, it was confirmed that in 910 nAChRs most of the amino acid residues that Similar experiments were performed using a second line the pore are also part of the TM2 domain from each mutation at position 17, including 9I17T and 10M17T. subunit. Interestingly, the leucine ring (position 9’, i.e, L9) These mutations also significantly enhanced the nAChR’s located in the middle of the ion channel has a key role in sensitivity to ACh. The results from these experiments gating [21]. Changes of this Leu residue to amino acids with involving the hydrophobic change at position 17’ to a Thr, hydrophilic side chains in either the α1, β1, δ, and γ subunits also confirmed that the receptor expressed had two α9 and from muscle nAChRs, conferred a major leftward shift to the three α10 subunits [23]. Plazas et al. [23] demonstrated that this dose-response curve for ACh, indicating that these structural stoichiometry was preferred and additionally fixed, changes produce hypersensitivity to ACh [22]. As it was independently of the availability of 9 or 10 subunits, since demonstrated for the 7 nAChR, changing Leu to Thr increasing the ratio of V13T9 to wild-type 10 subunits residues in the 910 nAChR resulted in a mutated receptor from 1:1 to 4:1 or increasing the ratio of V13T10 to with significantly increased activation potency and decreased wild-type 9 subunits from 1:1 to 4:1 increased the potency rate of desensitization for ACh [21]. Other substitutions at L9 for ACh. Thus, within this range of cRNA available, 910 in the α9α10 nAChR had similar effects as observed in the nAChRs preferentially assemble in a stoichiometry of two α9 7 nAChR. However, the substitution of valine at position and three α10 subunits [23]. 13’ resulted in a more pronounced increase in sensitivity to ACh than that elicited by the substitution at position 9’ [21]. Importance of the cytoplasmic domain in clustering and Indeed, when compared to other similar (hydrophobic to functional interactions of nAChRs hydrophilic) side chain mutations in TM2, the mutation of Val to Thr at position 13’ (i.e., V13T) conferred the largest The cytoplasmic domain between TM3 and TM4 is a key increase in sensitivity to ACh in the 910 nAChR [21]. molecular determinant for the selective subcellular targeting Thus, this mutation was chosen to investigate the of specific nAChRs subtypes. It is clear that different nAChR stoichiometry of the α9α10 nAChR expressed in Xenopus subtypes co-exist in various cells, and that they may be oocytes by determining the number of components in clustered into or specifically excluded from discrete domains dose-response curves obtained in experiments with receptors in those cells or even within individual synapses. For assembled from combinations of wild-type and mutant example, the 3 subunits are found clustered exclusively at subunits. The dose-response curve to ACh for nAChRs postsynaptic sites in neurons of the ciliary ganglion. Studies of containing the 9V13T mutant coexpressed with the the distinct localization of chimeric receptors in which the wild-type 9 and 10 subunits was demonstrated to have cytoplasmic domain sequence had been substituted three components. One component corresponds to the demonstrated that the cytoplasmic domain was sufficient to expected EC50 value for the 910 nAChR, whereas the direct the localization of the 3 subunit to specific [24, other two components with lower EC50 values reflect the sub-synaptic domains in chicken ciliary ganglion neurons incorporation of either one or two 9V13T mutated subunits 25]. In contrast, the 7 subunits, which are expressed at in the nAChR complex [23]. In the same manner, comparable levels in the same neurons, are specifically coexpression of the 10V13T mutant with the wild-type 10 targeted to extrasynaptic regions [25]. The cytoplasmic domain and 9 subunits resulted in dose-response curve to ACh with of the 4 subunit interacts with the chaperone protein 4 components, confirming the presence of three 10 subunits 14-3-3eta and this interaction regulates the association of the

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4 subunit with scaffolding proteins and ultimately outer hair cells), in the basal pole of the outer hair cells, and in determines its sub-cellular localization [26]. Most of the efferent synaptic contacts below the basal region of the outer sequence divergence found among nAChR subunits is found hair cells. Because association between the 10 subunit and in this domain, reflecting the domain’s critical role in prosaposin would require their coexpression in the outer hair targeting specific subcellular regions and also in the cells, and immunofluorescence had failed to reveal prosaposin interaction with other proteins. The best amino acid alignment expression in these cells, the expression of prosaposin was of the cytoplasmic domains from the 7, 9, 10, and 3 further investigated in microdisected cells by RT-PCR. The subunits from human (H), mouse (M), chicken (C), and rat (R) results of these experiments agreed with the (Fig 1), demonstrates that there is significant sequence immunofluorescence results, where prosaposin mRNA was conservation across species within each subtype, but only five more abundant in supporting cells, but the results also amino acids are conserved across the different subtypes, confirmed that prosaposin mRNA was present in the outer namely P333, W335, L431, I431 and A431, in the H9 hair cells and thus may serve as a binding partner to the 10 sequence (white letter on black background; Fig 1). A recently subunit, as indicated by the yeast-two hybrid assays. Intense published alignment of 57 9 sequences also demonstrated a prosaposin label was observed in the cytoplasm of the high conservation of these five amino acids. The residues Scarpa’s ganglion neurons and around the nucleus of the P333, W335 and I431 are conserved in all sequences, L is vestibular hair cells of the ampulla and utricle. Less intense conserved in 40 sequences, while the remaining 17 sequences label was apparent in the saccular hair cells [30]. have I instead in this position, and only three sequences have A432 substituted by V, S, or T [27]. It is not known if there is Prosaposin knockout mice exhibited normal hearing functional significance for the high conservation of those five thresholds only when very young mice were used. In 22 days amino acids amongst the nAChR subtypes. However, in the old mice, significant rapidly progressing hearing loss was midst of the sequence divergence of the cytoplasmic domain evident in the homozygotes mice lacking prosaposin, while amongst subtypes the conservation of these five amino acids the wild-type and heterozygous prosaposin-expressing is notable. littermates retained normal hearing. By one month of age, the knockout mice were deaf. This was accompanied by Yeast-two hybrid screening is a molecular biology abnormalities in the organ of Corti innervation, and approach that has been used extensively to study hypertrophy in the inner hair cell and the Deiter cells regions. protein-protein associations. To discover proteins that interact Labeling for neuronal markers neurofilament and with the α9α10 nAChR in auditory hair cells, Akil et al. [28] synaptophysin specifically for efferent endings was reduced in used this approach and the cytoplasmic domains of α9 and α10 the immature organ of Corti when compared to the as ‘baits’. The cytoplasmic domains of the α9 and α10 immunofluorescence labeled observed in prosaposing subunits were selected as baits because these domains have expressing littermates. However, after the onset of hearing, been shown to determine the localization of other members of the labeling of both of these markers was more intense and the nAChR family and because they are expected to have sites concentrated abnormally in the inner hair cell and Deiter cell of association with other proteins. Using the cytoplasmic regions, where cellular hyperplasia was observed. In mice domain of α9 as bait, no interacting proteins were identified. lacking prosaposin, loss of outer hair cells in the apex of the Using the cytoplasmic domain of α10, Akil et al. [28] identified cochlea was also prevalent by one month of age [29]. a protein, prosaposin, which had not been described in the cochlea [28, 29]. Prosaposin is a precursor of four glycoprotein Because 910 nAChRs are expressed in the vestibular activators of lysosomal hydrolases (saposin A, B, C and D). sensory organs, the effects of prosaposin deletion in vestibular Systematic deletion analysis and yeast growth assays revealed function was evaluated. Using four tests, the air righting that the prosaposin site for binding to α10 was localized to a reflex, contact righting reflex, swimming test, and 40 amino acid region in the carboxy termini, corresponding to circling/rearing frequency test, which are routinely used to a domain in saposin D [29]. This domain of saposin D had not evaluate vestibular function in mice, measurements of been previously found to interact with other proteins. vestibular function demonstrated significantly impairment in prosaposin knockout mice [30]. Consequently, it is clear that Immunofluorescence using an antibody that recognizes prosaposin, or one of its mature cleaved products, saposins A– both prosaposin and its cleaved product, saposin D, revealed D, is very important for normal hearing and also vestibular labeling throughout the immature cochlear sensory epithelia functions. Prosaposin, or one of its mature cleaved products, in very young mice (one and five days old). In 10 days old and saposins A–D is essential for the maintenance of innervation older mice, the labeling was detected in the inner hair cells, within the mammalian organ of Corti, and is specifically inner pillar cells, Deiter cells (supporting cells underneath the required for normal development of the efferent connections. Prosaposin

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Figure 2. Diagram of the nAChR and SK2 channels located post-synaptically to efferent fibers in auditory outer hair cells. In high frequency regions of the cochlea, in addition to the SK2 channels shown, BK channels (large conductance Ca2+ and voltage-activated channels), are also expressed [48, 49]. The ACh-induced conductance results in efferent modulation of the cell’s excitability. The nAChR is thought to be a pentamer of two 9 and three 10 subunits (for clarity only one subunit of each type is shown). The SK2 channel consists of four pore-forming subunits each bound to one calmodulin molecule through a calmodulin binding domain located at their carboxy termini (only one SK2 subunit and one calmodulin molecule are shown). ablation has specific effects on efferent functions in the demonstrated in various tissues and diverse cell types. In auditory and vestibular epithelia, and this functional most cases, however, their functions are the subject of impairment may be due to lack of direct interaction with 10 intense investigation and still unclear. Initial in situ nAChR subunit, as indicated by the Y2H results [29]. hybridization studies demonstrated the expression of the α9 subunit in E16 rat embryonic pituitary, nasal epithelium, Prosaposin has been shown to function as a neurotrophic tongue, sternohyoid muscle, adult rat pituitary, and organ of [4] factor and also may bind to Go-coupled receptors. A possible Corti . In the rat, the expression of both α9 and α10 association of 910nAChRs with G-proteins through mRNAs was demonstrated in neurons of the dorsal root prosaposin and the functional implications of such ganglia using in situ hybridization. Additionally, interactions warrant further research given the atypical immunoreactivity experiments demonstrated α10 expression pharmacology of these nAChRs in hair cells, which have been at the protein level in the dorsal root ganglia, and the demonstrated to exhibit mixed muscarinic and nicotinic coexpression of the α9 and α10 subunits was further properties [23]. Indeed, substantial evidence points to confirmed by RT-PCR and western blotting in this ganglia involvement of GTP-binding proteins in the ACh-induced [31]. Indeed, α9α10 nAChRs were detected in all neurons of response of the outer hair cells [20, 61-63]. ACh-induced currents the dorsal root ganglia, not only in the nociceptor neurons. of isolated outer hair cells were significantly reduced when Thus, the authors concluded that α9α10 nAChRs are likely to G-protein inhibitors were dialyzed through the patch pipette fulfill a general function in this ganglia not necessarily [20] [31] . Specifically, Giα2 immunoreactivity was detected in the related to nociception . [62] hair cells of the mouse cochlea . A Giα2 cDNA, similar to the mouse macrophage Giα2, was cloned from a mouse It has been additionally proposed that in epidermal, basal cochlea cDNA library, and a second cDNA clone, had also and follicular cell layers from the skin, ACh is involved in been obtained from the auditory sensory epithelia (GenBank the transition of differentiating keratinocytes from the accession #s AK157998, S71213). stratum granulosum to the stratum corneum [32, 33]. Both in vitro an in vivo models of inactivation of α9 nAChR-induced Functions of α9α10 nicotinic acetylcholine receptors signaling using antagonists or RNA interference of keratinocyte as well as experiments in knockout mice The expression of α9 and α10 subunits has been delayed wound healing, and reduced colony scattering and

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Neurotransmitter 2016; 3: e1298. doi: 10.14800/nt.1298; © 2016 by Ana E. Vázquez http://www.smartscitech.com/index.php/nt cell outgrowth from the colony. The α9 nAChR was shown Calcium-activated K+ channels are present in all neurons to influence the gene expression of adhesion molecules, and mediate the after hyperpolarization following action suggesting that α9 nAChR signaling regulates the assembly potentials. They are involved in many physiological and disassembly of adhesion complexes, both of the processes including neurosecretion and smooth muscle tone, cell-extracellular matrix and cell-cell types, through as well as modulating the action potential shape, and spike transcriptional, translational and postranslational frequency adaptation. There are three main subfamilies of mechanisms involving structural and effector molecules K(Ca2+) channels based on their electrophysiological, important in cell shape, cytoplasm motility, and adhesive pharmacological phenotypes, and molecular properties. The properties of keratinocytes [34]. large-conductance Ca2+- and voltage-activated K+ channels (BK) are sensitive to block by charybdotoxin (CTX) and PCR, western blot and immunohistochemical studies iberiotoxin, the intermediate-conductance KCa channels are demonstrated the expression of the α9 and α10 subunits in inhibited by CTX and clotrimazole, and the small populations of T-lymphocytes, however, no response to ACh conductance KCa channels (SK) are sensitive to apamin, was detected from any of these cells [35]. It is also known that scyllatoxin, and dequalinium chloride. most of the nAChRs containing α9 and α10 subunits are expressed in human and mouse bronchial epithelia [36]. The In rat organ of Corti preparations, presynaptic function and the mechanisms of action of these receptors in depolarizations were shown to induce an inhibitory these cells remain to be elucidated. hyperpolarization of the outer hair cells [43]. This response was blocked by 100 nM strychnine, an 9 nAChR blocker, and Contrary to this, in the auditory and vestibular sensory by 1 µM dequalinium, an SK channel blocker [43, 44]. Thus, organs it is clear that 9/10-containing nAChRs are crucial nAChRs from the outer hair cells are thought to be as efferent targets for the feedback modulation of the heterooligomers composed of 9 and 10 subunits [3, 28]. excitability of the hair cells. 9 mRNA has been detected Although there is significant sequence homology between using in situ hybridization in the chick basilar papilla [37] and the 9 and 10 subunits, and the 9 nAChR forms in auditory hair cells from rats and guinea pigs [4, 38, 39]. The homomeric channels when expressed alone in Xenopus 10 subunit has been cloned from a rat cochlea cDNA oocytes [4], when the 10 RNA is injected by itself no library, and transcripts were detected using in situ homomeric ACh-activated channels were expressed. hybridization in auditory and vestibular hair cells in the rat [3, However, co-injection of the 9 and 10 subunits resulted in 40]. Efferent cholinergic fibers from the brainstem enter the the expression of ACh-sensitive channels with 100-fold inner ear forming synapses on the basolateral pole of sensory larger current amplitude and thus significantly different from hair cells and modulating their excitability. This modulation 9 homomeric receptors. The 9 subunit was then suggested is essential to our sense of hearing since it sharpens to be essential in the trafficking of the 10 subunits to the frequency selectivity and contributes to protection from plasma membrane [45]. Additionally, when the 9 and 10 [6, 40] excessive noise . The molecular basis of this modulation where co-expressed, the properties of the receptors has been the topic of extensive controversy in the last few resembled the native hair cell receptors, indicating a direct years. In contrast to all other nAChRs which support interaction between the subunits. Other lines of evidence also excitatory neurotransmission in auditory hair cells, confirmed the interaction between these subunits. For 2+ agonist-evoked nAChR channel opening results in Ca example, in immunoprecipitation experiments of cochlear influx and brings about cellular hyperpolarization. Figure 2 protein extracts using 9 and 10 specific antibodies it was provides a diagram of the major conductances involved in demonstrated that each subunit may precipitate the other one the process of nAChR activation. Physiologically, this [46]. Additionally, when the extracts were prepared from hyperpolarization of the hair cells results in a reduction of the transgenic mice engineered to overexpress the 9 subunit, movement of the basilar membrane thus, dampening hearing larger quantities of the 9 and also of 10 subunits were sensitivity [6]. The α9 and α10 subunits are key molecular constituents mediating efferent effects on the hair cells [41, 42]. recovered in the immunoprecipitated complexes compared to extracts from wild-type mice [43]. Noticeable, in mice, there is Recent studies utilizing α9/α10 knockout mice and mice lacking expression of SK2, a K+ channel that is functionally a broad variability of the 9 expression levels in the hair associated with α9/α10 nAChRs, provided additional cells and this was found to be correlated with the strength of evidence for the essential role of these receptors in the the protection from noise damage provided by the efferent [47] development and modulation of the hair cell’s efferent system . This direct correlation between 9 protein levels synaptic function [24, 25]. Their co-localization – or clustering and the strength of protection from acoustic damage – is critical for the influx of Ca2+ and immediate gating of a conferred by the efferent system supported a direct central Ca2+-dependent K+ conductance [43]. role for 9 in the efferent system and has been recently

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Neurotransmitter 2016; 3: e1298. doi: 10.14800/nt.1298; © 2016 by Ana E. Vázquez http://www.smartscitech.com/index.php/nt confirmed by studies of knockin mice (discussed below) [47]. ACh-induced currents recorded from immature inner hair cells of L9T/wt and L9T/L9T mice displayed higher The immature sensory hair cells produce spontaneous sensitivity to ACh and decreased desensitization rates electrical activity and the final differentiation of the mature compared to the ones recorded from wild-type mice, inner hair cells seems to require specific patterns of resembling the characteristics of the reconstituted L9T spontaneous action potential during a critical period. The α9 receptor in Xenopus oocytes [21, 53]. In immature inner hair is required for the development of inner hair cell synapses cells following neurotransmitter release, spontaneous and specifically for the Ca2+-induced vesicle fusion at the inhibitory postsynaptic currents (IPSC) reflect opening of the inner hair cell synaptic machinery. nAChR and subsequent gating of the associated Ca2+- activated K+ channels. These currents in L9T mutant mice The 910 nAChR exhibits marked differences in Ca2+ were more than double in magnitude, when compared with permeability across species. Mammalian recombinant and currents in wild-type mice. Additionally, in these cells the native 910 nAChRs have relative Ca2+ to monovalent spontaneous IPSC, as well as IPSC evoked by 40 mM K+ cation permeability ratios close to or higher than 10 [14, 50-52]. lasted much longer. The activation of K+ currents In contrast to this, the relative Ca2+ to monovalent cation concomitant to AChR activity was not affected by the permeability ratio for the chicken recombinant 910 mutation. Although electrophysiological studies in mature nAChR is much lower, a ratio of 2, which is similar to other outer hair cells are more technically challenging that in nAChRs that do not contain the 910 subunits (50). The immature inner hair cells, the effects of the L9T mutation authors conclude that the evolutionary processes that were were also studied on the mature outer hair cells. The responsible for the increased Ca2+ permeability of ACh-evoked response in outer hair cells from mice carring mammalian 910 nAChRs were specific to the mammalian the L9T mutation was longer-lasting and more sensitive to lineage and happened concomitant with other adaptations ACh than this response in outer hair cells from wild type that conferred high frequency hearing to mammals [27, 48-50]. mice. In general, all the properties confirmed that outer hair cells expressed the same L9T-containing nAChR as the one Insights from studies in transgenic mice expressed in the immature inner hair cells.

Following the characterization of site-directed mutant Interestingly, the hearing thresholds were 5-15 dB higher nAChRs expressed in Xenopus oocytes, and the confirmation in both, the double mutant as well as the heterozygous when of the critical role of TM2 amino acids in the gating process compared to the wild-type mice. This mutation strengthened at 910 nAChRs, the construction and study of the cochlear suppression in vivo demonstrating the central role corresponding knockin mice was invaluable to advance our of 9 nAChRs in the medial efferent system. The study of knowledge of the native receptor. The substitution of Leu to this mutation also demonstrated that this system protects [53] Thr at position 9’ in the TM2 from the α9 subunit (9 L9T) from damage elicited by excessive noise . when coexpressed with the α10 subunit in Xenopus oocytes Phenotype of α9 knockout mice (α9-/-) displayed decreased EC50 and decreased rate of desensitization to ACh [21]. The introduction of this mutation in the mouse produced wt/L9T heterozygous as well as Mice lacking α9 expression were viable, had no noticeable L9T/L9T double mutants which had no differences in life abnormal phenotype and their cholinergic olivocochlear [42] span, seemed to age and reproduce normally, had no notable bundle was normal . Since the 910 nAChR was though behavioral phenotype, and the cochlear was normal. to play a major role in efferent neurotransmission, various Additionally, the mRNA level of 9 L9T mutant nAChRs aspects of inner ear function and specifically of the efferent was similar to that for the 9 in wild-type mice, and there pathway were studied in the 9 knockout mice. For example, were no differences in the expression levels of α10 or SK2 measurements of cochlear compound action potentials report either. It was also demonstrated that the capacitance and the the activity of cochlear afferent fibers and thus reflect the voltage-dependent K+ currents of the auditory hair cells were function of the inner, outer hair cells, and afferent neurons. not affected by the mutation. This comprehensive evaluation All α9 knockout mice had normal compound action potential of the phenotypes in the knockin homo- and hetero-zygous thresholds. Distortion product otoacoustic emissions mice provides confidence that the effects of the L9T (DPOAEs) are emissions produced by the inner ear when it is mutation on hearing physiology are due to the direct role of challenged with two tones. These emissions specifically 9 on ACh neurotransmission in the cochlear hair cells and reflect the function of the outer hair cells. There were no to the altered channel properties, and not to secondary effects differences in the DPOAEs in 9 knockout mice when or artifacts of the mutation. compared to wild-type mice. Thus, the lack of the α9 subunit affects neither the normal inner and outer hair cells, nor

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Neurotransmitter 2016; 3: e1298. doi: 10.14800/nt.1298; © 2016 by Ana E. Vázquez http://www.smartscitech.com/index.php/nt afferent neuron functions. However, functional deficits cells from the wild-type and heterozygous littermates, specific to the efferent system were revealed. In wild-type, α10-expressing mice, inward currents in the range of 200- α9-expressing mice, the activation of the efferent fibers 500 pA were routinely recorded under the same conditions either electrically or acoustically reduces the amplitude of the [56]. compound action potential and of the DPOAEs, whereas in the α9 knockout mice, this activation had no effect either on All α10 knockout mice had normal baseline hearing, and the compound action potential or on the DPOAEs [42, 54]. This normal DPOAEs. However, the efferent innervation of the confirmed that nAChRs containing the α9 subunit play an organ of Corti seemed to be disorganized, and in general essential role in the efferent feedback mechanism. resembled the abnormal innervation in the organ of Corti of the α9 knockout mice [42, 56]. The α10 mouse had abnormal In addition to the lack of functional efferent effect due to efferent innervation in the sense that there were less number the lack of the α9 subunit, studying 9 knockout mice also of presynaptic terminals in the outer hair cells and on average revealed a reduced and abnormal innervation of the organ of the terminals were larger in size, and mostly single, instead Corti. In the organ of Corti, the innervation of the outer and of in clusters, as they are normally found in the inner hair cell areas corresponds to the projections of lateral α10-expressing mice. Functionally, the α10 deletion in vivo and medial components of the olivocochlear efferent bundle. demonstrated that α10 was essential for the efferent effects In wild-type mice, presynaptic efferent terminals contact the on hearing function [56]. basal pole of the outer hair cells as clusters of up to five terminals per cell, and there is also a profuse innervation Potential role of α9α10 nAChRs as therapeutic targets consisting of smaller terminals contacting inner hair cells or neurons of the spiral ganglia under the inner hair cell region The potential for α9α10 nAChRs to be used as therapeutic [42, 55]. The effect of the lack of α9 expression on the integrity targets is being intensively explored. α9α10 nAChRs seem to of these terminals was studied using two different markers of hold promise as attractive pharmacological targets partly the presynaptic sites, synaptophysin and vesicular because they have not been found assembled with other acetylcholine transporter. Immunostaining with both of these nicotinic subunits, and because of their distinct markers demonstrated that in α9 knockout mice there were a pharmacological profile when compared to that for other lesser number of presynaptic terminals in the outer hair cells nAChRs. For example, various nAChRs bind α-conotoxins and that they were larger in size, and mostly single, instead (α-CTx’s) with high selectivity. These are small disulfide of in clusters, as they are normally found in α9-expressing rich peptides found in the venom of marine snails, and there mice. Additionally, the innervation of the inner hair cell area is a vast diversity of them since there are around 700 species seemed to lack the terminals closely associated with the inner of these snails [57]. Notably, α9α10 nAChRs displayed a hair cells. Thus, the lack of hair cell response due to the α9 260-fold higher affinity than α7 nAChRs for a recently deletion has an effect on the maturation of the efferent fibers discovered α-CTx, α-CTx PeIA. α-CTx PeIA thus may be and less (non-productive) contacts are established. Thus, the useful to discriminate physiological processes mediated α9 deletion precluded the normal development of the efferent through α9α10 or α7 nAChRs in tissues where both receptors fibers in the organ of Corti of the knockout mice [42, 54]. maybe expressed [58]. Additionally, the α-CTx’s RgIA and Vetter et al. [42] proposed mechanistic hypotheses to explain Vc1.1 selectively block α9α10 nAChRs but no other nAChRs how does the α9 deletion may bring about this effect. [59]. These α-CTx’s are effective pain killers, and also seem to accelerate functional recovery from nerve injury [57, 59, 60]. Phenotype of α10 knockout mice (α10-/-) α-CTx RgIA and Vc1.1 bind to α9α10 nAChRs with nanomolar affinities and are thus powerful selective As it was the case for the α9 subunit, Vetter et al. [56] antagonists of α9α10 nAChRs. α-CTx RgIA reduced acute demonstrated that the α10 subunits are also required for the pain and produced a sustained analgesic effect in the chronic expression of functional nAChRs in auditory hair cells and constriction nerve injury model of neuropathic pain in rats for normal development of the efferent fibers in the organ of [61]. Additionally, because of their unique roles in the Corti. Although it had been previously shown that α9 development and modulation of the sensory end organs of nAChRs form functional homomeric receptors when hearing and balance, the potential of α9α10 nAChRs as expressed alone in Xenopus oocytes [3, 4], and that the α10 pharmacological targets for specific pathologies of the deletion had no effect in the α9 transcript levels, no currents auditory and vestibular systems is being explored [reviewed were recorded in response to ACh applications in inner hair recently [62]]. There also seems to be a niche for α9α10 cells from mice lacking α10 expression. There was no nAChRs as therapeutic targets for pathologies of the skin, response even at high concentrations (i.e., 100 µM or 1 mM) bronchia and even in cancers; however, the understanding of of ACh in hair cells from α10 knockout mice, while in hair the specific roles of α9α10 nAChRs in these tissues is still

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Neurotransmitter 2016; 3: e1298. doi: 10.14800/nt.1298; © 2016 by Ana E. Vázquez http://www.smartscitech.com/index.php/nt being elucidated. Opin Neurobiol 1996; 6:514-519. 6. Guinan JJ, Jr. Olivocochlear efferents: anatomy, physiology, Summary function, and the measurement of efferent effects in humans. Ear Hear 2006; 27:589-607. α9α10 nAChRs define the most phylogenetically distant 7. Guinan JJ, Jr., Warr WB, Norris BE. Differential olivocochlear subfamily of nAChRs. Notably, in contrast to all other projections from lateral versus medial zones of the superior cholinergic neurotransmission which serve excitatory olivary complex. J Comp Neurol 1983; 221:358-370. functions, cholinergic neurotransmission mediated by α9α10 8. Elgoyhen AB, Katz E. The efferent medial olivocochlear-hair cell nAChRs is inhibitory. α9α10 nAChRs have a much higher synapse. J Phys, Paris 2012; 106:47-56. 2+ 2+ selectivity for Ca and serve as Ca source for the 9. Fuchs PA, Murrow BW. 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