Ligand-gated ion channels are found throughout the brain and body, where they mediate fast synaptic transmission. These ligand-gated ion channels include the 5-HT3, glycine, GABA type A and C, and nicotinic acetylcholine receptors. These receptors are also occasionally referred to as the Cys-loop receptors, since all have the highly conserved feature of a pair of cysteine amino acids separated by 13 residues and linked by a disulfide bridge (Gotti and Clementi,2004).
The nicotinic acetylcholine receptors (nAChRs) represent an important subcategory of these ligand-gated ion channels; the nAChRs are multisubunit, neurotransmitter-gated receptors of the cholinergic system and filogenetically one of the oldest and most important pathways within the nervous system. These ionotropic receptors are responsible for mediating the effects of the neurotransmitter acetylcholine (ACh) and are also the principal target of the biologic compound nicotine, which typically, though not always, mimics the effects of acetylcholine on the nAChR (Lukas,1995). Classically, nAChRs have been divided into two groups: muscle receptors, found at the skeletal neuromuscular junction, where they mediate neuromuscular transmission, and neuronal receptors, found throughout the central and peripheral nervous system (Gotti and Clementi,2004). This distinction between neuronal and nonneuronal subtypes does not always hold up, however, as the neuronal receptors have been identified in a variety of nonneuronal tissues (Nguyen et al.,2000; Peng et al.,2004)
The nAChRs play crucial physiologic roles throughout the central and peripheral nervous system. They regulate neurotransmitter release, cell excitability, neuronal integration, and networking and are intimately involved in such important functions as sleep and arousal patterns, fatigue, hunger, anxiety, and pain processing (Lindstrom,1997; Changeux and Edelstein,2001; Hogg et al.,2003; Gotti and Clementi,2004). Dysfunction of nAChRs are implicated in a variety of human diseases and health conditions, including epilepsy, tobacco addiction, schizophrenia, myasthenia gravis, and some forms of skin disorders (Lindstrom,1997; Nguyen et al.,2000). Their function most likely extends beyond the boundaries of neural function since they have also been identified in such diverse tissues as lymphocytes and keratinocytes (Nguyen et al.,2000; Peng et al.,2004).
Since their initial discovery in the auditory system a little more than a decade ago (Elgoyhen et al.,1994), the role of nAChRs in normal hearing is slowly being elucidated. The remainder of this article will summarize current knowledge regarding nAChR structure and function within the mammalian auditory system, highlighting current research trends in this area.
ACETYLCHOLINE RECEPTOR STRUCTURE
ACh acts on two classes of receptors, nicotinic and muscarinic, each demonstrating unique functional, pharmacologic, and molecular properties. All nicotinic acetylcholine receptors are ligand-gated ion channels and are pentamers composed of homologous protein subunits, each subunit spanning the plasma membrane four times (Sargent,1993). In contrast, muscarinic acetylcholine receptors do not form channels by themselves, but rather are members of a class of receptors coupled to a G-protein, formed by a single polypeptide chain spanning the plasma membrane seven times (Bonner et al.,1987). All the nAChR subunits have been identified by molecular cloning techniques and numbered in the order in which they were identified. There are 10 α-subunits (α1–α10), 4 β-subunits (β1–β4), and γ-, δ-, and ϵ-subunits, all with shared homology. Various combinations of subunits form subtypes of the acetylcholine receptor.
One of the more thoroughly studied nAChRs, due to their involvement in the autoimmune disease myasthenia gravis (Drachman,1994), is the muscle receptors, containing the nAChR subunit α1. These nAChRs are made up of five subunits arranged around a central pore. In adult vertebrate muscle, the stoichiometry of the receptor is (α1)2-β1δϵ; however, the pentameric composition is tightly regulated such that the clockwise order is consistently α1-ϵ-α1-β1-δ (Mishina et al.,1986).
In contrast, the neuronal receptor subunits α2–10 (though, again, these receptors are also found within nonneuronal tissues) are less tightly constrained stoichiometrically as compared to the muscle nAChR. Most receptors contain a single type of α-subunit and a single type of β-subunit, though some receptor complexes have been demonstrated in Xenopus expression systems with two or three types of subunits (Boorman et al.,2000). For example, nAChR subunits α2–α4 will only form functional receptors when combined with either β2 or β4. The nAChRs that bind α-bungarotoxin, α7–α9, are somewhat unique in that they can be functionally expressed as homomeric receptors by themselves without a β-subunit within in vitro mammalian and Xenopus expression systems (Boulter et al.,1987; Deneris et al.,1988; Wada et al.,1988; Elgoyhen et al.,1994). Another distinguishing feature of the α7–α9 nAChRs are their high calcium permeability (Seguela et al.,1993; Lindstrom et al.,1998).
The ligand-binding site for ACh is created at the interface of the α-subunit and its immediate neighbor in the pentameric structure (Corringer et al.,2000). The variations in subunit composition therefore helps to explain the varying pharmacology of this diverse group of nAChRs (McGehee and Role,1995; Hogg et al.,2003). The precise number of ACh binding sites in each receptor complex remains speculative, though the receptors with two α- and three β-subunits are presumed to have two.
The secondary structure of the nAChR has been elucidated through a variety of methods (Fig. 1). Each α-subunit consists of an N-terminal extracellular domain that contains the ACh binding region. There are four hydrophilic membrane-spanning domains (often labeled as M1–4) and an intracellular loop between M3 and M4. It is variations in this intracellular loop region that largely accounts for the diversity of the α-subunits. This intracellular loop also frequently contains one or more phosphorylation sites. Each functional receptor is comprised of five subunits to form a circular ring, the central pore of which is the ion channel of the receptor (Toyoshima and Unwin,1988,1990; Sargent,1993). Evidence suggests that ACh binding to the receptor leads to a conformational change, which entails a 15–16° rotations of the inner pore-facing parts of the α-subunits, which then acts as the trigger that opens the gate within the membrane-spanning pore (Unwin et al.,2002).
NICOTINIC ACh ACTIVITY IN AUDITORY SYSTEM: EFFERENT INHIBITION
To date, the primary role identified for nAChR function within the inner ear is related to efferent auditory neuronal activity. Mechanosensory hair cells of the organ of Corti transmit information regarding sound to the central nervous system by way of peripheral afferent neurons. The central nervous system provides feedback and modulates the afferent stream of information through efferent cholinergic neurons, which arise in the brainstem and comprise the olivocochlear pathway (Rasmussen,1946; Galambos,1956; Wiederhold and Kiang,1970; Guth et al.,1976). This efferent system can be subdivided into the lateral efferents that terminate on the afferent dendrites innervating the inner hair cells (IHCs) and the medial efferents that terminate directly on IHCs during development, and later directly on the outer hair cells (OHCs; Fig. 2) (Liberman and Brown,1986; Warr et al.,1986; Simmons,2002). Activation of the efferent system, either by sound or by direct electrical stimulation, is inhibitory and results in broader tuning and reduced sensitivity of the cochlear afferents (Galambos,1956; Wiederhold and Kiang,1970; Klinke and Galley,1974; Eybalin,1993). Though still under debate, this efferent inhibition may function to extend the dynamic range of afferent fibers (Winslow and Sachs,1987), is believed to improve the ability to detect signals in noise (Huang and May,1996; Hienz et al.,1998), and may be involved in mediating aminoglycoside-induced ototoxicity (Capps and Duval,1977; Aran et al.,1999). There is also a growing body of evidence that this efferent pathway also provides some protection against noise-induced cochlear injury (Liberman,1988). The strength of this efferent system and the physical number of the outer hair cell efferent receptors present in the inner ear both have been directly correlated with individual susceptibility to noise-induced hearing loss, whereas overexpression of the outer hair cell efferent receptor has been shown to be protective against noise-induced hearing loss (Maison and Liberman,2000; Luebke and Foster,2002; Maison et al.,2002).
Even before the discovery of the nAChR responsible for cochlear efferent inhibition, it was widely held that ACh was the primary efferent neurotransmitter in the inner ear (Guth et al.,1976; Kujawa et al.,1992; Eybalin,1993). Within the cochlea, ACh is generally accepted as the main neurotransmitter between the efferent olivocochlear system and the outer hair cell (Kujawa et al.,1992). While other efferent neurotransmitters have been described, their function remains to be determined (Guth et al.,1976; Eybalin,1993).
Much of our understanding of efferent physiology within the inner ear has emerged from studies of the hair cells of cold-blooded vertebrates (Flock and Russell,1973) and by the application of acetylcholine (ACh) and its antagonists to hair cells isolated from birds and mammals. Early studies in isolated chick cochlear hair cells demonstrated a biphasic inhibitory response after the application of ACh (Fuchs and Murrow,1992a, Fuchs and Murrow,1992b). This response was mediated by a ligand-gated cation channel, with subsequent activation of a calcium-activated potassium channel. Results from these studies and others have led to a two-channel hypothesis of hair cell inhibition, whereby ACh causes the influx of calcium through an ACh-gated cation channel, leading to the opening of a calcium-activated potassium channel (Fig. 3) (Fuchs and Murrow,1992b; Martin and Fuchs,1992). Additional experiments in such divergent species as the turtle (Art et al.,1984,1985) and guinea pig (Blanchet et al.,1996; Evans,1996) auditory end organs demonstrating similar ACh-mediated inhibitory responses, strongly suggesting that cochlear hair cell inhibition is a highly conserved process in all vertebrates. An unusual feature of this inhibitory response in hair cells is that it can be blocked by both nicotinic and muscarinic antagonists (Bobbin and Konishi,1974; Shigemoto and Ohmori,1991; Fuchs,1992a,1992b; Housley et al.,1992; Kakehata et al.,1993; Erostegui et al.,1994; McNiven et al.,1996). It was in fact this dual nicotinic-muscarinic profile that ultimately led to the identification of the two novel nAChR subunits within the inner ear (Elgoyhen et al.,1994,2001; Lustig et al.,2001).
MOLECULAR BASIS OF OUTER HAIR CELL INHIBITION: nAChR α9α10
The first clues to the identity of the cochlear efferent nAChR arose during functional expression studies of the homomeric nAChR α9. These expression studies in Xenopus oocytes demonstrated responses that were remarkably similar to electrophysiologic responses previously identified in isolated outer hair cells (Elgoyhen et al.,1994). However, there were some physiologic differences between isolated OHCs and expressed homomeric α9 receptors that did not fully support this notion, including differing current-voltage relationships, Ca2+ sensitivity, and desensitization kinetics (Blanchet et al.,1996; Dulon and Lenoir,1996).
Subsequent coexpression studies of a heteromeric receptor composed of the α9 subunit together with a newly identified α10 receptor subunit demonstrated a physiology more closely matching the native hair cell response (Elgoyhen et al.,2001; Sgard et al.,2002). As compared to expressed homomeric α9 receptors, the heteromeric α9α10 receptors displayed faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular calcium ions, a pharmacology much more closely matched to isolated OHC responses (Elgoyhen et al.,2001). Since these studies, a variety of physiologic, molecular, and immunohistologic studies in animals and humans have further strengthened the assumption that the hair cell cholinergic receptor is a heteromer of the α9 and α10 subunits (Lustig et al.,2001; Lioudyno et al.,2002; Morley and Simmons,2002; Sgard et al.,2002; Simmons,2002; Weisstaub et al.,2002)
The α9α10 receptor is unique from the other nAChRs in a variety of ways. As already noted, it has a highly characteristic mixed nicotinic- and muscarinic-sensitive pharmacology. Another interesting feature of the α9α10 receptor is its similarity to 5-HT3 ligand-gated ion channels. Tropisetron and ondansetron block ACh-evoked currents in α9α10-injected Xenopus oocytes, while serotonin blocks recombinant α9α10 receptors in a noncompetitive and voltage-dependent manner (Rothlin et al.,2003). The homomerically expressed α9 receptor is also reversibly and noncompetitively (with respect to ACh) blocked by aminoglycoside antibiotics, suggesting that some of the ototoxic effects of these drugs may be mediated by the efferent auditory system (Rothlin et al.,2000). The α10 subunit also has somewhat of a unique role as compared to other nAChR α-subunits. To date, the α10 receptor has only been shown to form a functional receptor when combined with α9; coexpression α2–6 or β2–4 does not yield a functional receptor in Xenopus oocyte expression systems (Elgoyhen et al.,2001). Thus, in many respects, the α10 receptor behaves as a β-receptor in other nAChRs, modulating the pharmacology of the α9 receptor. This is supported by studies that show that α10 has only been found expressed in association with α9 and never by itself in a variety of tissues studied, including lymphocytes and dorsal root ganglion neurons (Fig. 4) (Lustig et al.,2001; Lips et al.,2002; Peng et al.,2004). The one exception to this rule is in mature inner hair cells, which express α9 but not α10 (though the immature IHC does show expression of both subunits) (Elgoyhen et al.,2001; Morley and Simmons,2002).
A final interesting feature of this nAChR is the close genetic relationship of the two subunits that comprise the functional receptor. Among other human nAChRs, α10 is most closely related to α9, having 53% identity at the amino acid level, followed by α7, with which it shares 42% identity (Lustig et al.,2001). It has no more than a 35% homology with the remainder of the human nAChRs α1–6. Thus, human α10 is most closely related to the nAChR subgroup α7–9. Phylogenetically, α7–10 are believed to be the earliest of the nAChRs, being the first to diverge from the common ancestral subunit that gave rise to all the nAChRs (Tsunoyama and Gojobori,1998). Of these, α10 appears to be phylogenetically the oldest and may explain its functional role similar to a β-subunit (Fig. 5) (Tsunoyama and Gojobori,1998; Elgoyhen et al.,2001; Lustig et al.,2001).
While an overwhelming majority of the studies of the nAChR α9α10 have been carried out in the OHC of the auditory system, there is some evidence that this receptor plays additional roles in other neuronal and nonneuronal systems. Both α9 and α10 have been identified in the dorsal root ganglion of the rat (Lips et al.,2002). The neuronal receptor subunits have also been identified in a variety of lymphocytes, including B- and T-cells (Peng et al.,2004), where they have been postulated to provide a basis for parasympathetic regulation of the immune system (Rinner et al.,1995). This idea is not without precedent, as the closely related neuronal nAChR subunit α7 has also been shown to be involved in regulating B-cell proliferation and in mediating some aspects of the inflammatory response (Wang et al.,2003; Skok et al.,2005). Lastly, nAChR α9 has been identified in keratinocytes and it has been postulated to be the immune target in the potentially fatal autoimmune mucocutaneous blistering disease Pemphigus (Nguyen et al.,2000,2004).
GENOMIC ORGANIZATION OF nAChR α9 AND α10
The nAChR α9 gene lies on chromosome 4p14-15.1 (Lustig and Peng,2002). The gene contains five exons, separated by four introns. The intron-exon splice junction sites correlate identically with those of the rat α9 gene and are nearly identical to those of the human α10 gene. Sequence promoter analysis reveals a number of potential regulatory elements, including several in common with the nAChR α10 gene; 36 possible transcriptional factor binding sites have been identified in the nAChR α9 gene promoter region. As with the nAChR subunits α10 and α7, the α9 gene is believed to have TATA-less promoter (Gault et al.,1998, Lustig et al.,2001; Lustig,2002). Several promoter hot spots have been identified that contained three or more overlapping putative regulatory elements, including from −79 to −47 (BARBIE, LEF-1, and PU-1), −194 to −167 (HMGIY, FL-1, MZF-1, VDR/RXR), −384 to −367 (CEBPB, AMEF-2, and STAF), −770 to −712 (LTR TATA, HOX-1, ATF, NF-1, and TAL-1/E47), and −875 to −864 (CEBPB, MEF-2, and HSF-1). One study has provided evidence that a Sox complex, between +66 and +69 formed by two adjacent Sox boxes, provides an initial scaffold that facilitates the recruiting of the transcriptional machinery responsible for α9 subunit expression (Valor et al.,2003).
The α10 gene has been localized to chromosome 11p15.5 (Lustig et al.,2001). The genomic structure of α10 includes six exons, separated by five introns. Exon-intron splice sites 3–5 identically correlate with those of rodent and human α9 (Elgoyhen et al.,1994; Lustig and Peng,2002). Splice sites 3 and 4 also appear highly conserved among all the human nAChR α-forms. Since both the α9 and α10 subunits combine to form the functional hair cell receptor, one would presume that there would be similar regulatory transcriptional elements in the promoter region. A comparison of the putative α9 and α10 promoter regions indicates several potential candidates, including AP-4 (activator protein 4), NFAT (nuclear factor of activated T-cells), MZF-1 (myeloid zinc finger factor-1), BARBIE (barbiturate-inducible element), VDR/RXR (vitamin D receptor RXR heterodimer site), AREB6 (Atp1a1 regulatory element binding factor 6), STAF (Se-Cys tRNA gene transcription activating factor), and GATA-1 (GATA-binding factor 1) (Lustig and Peng,2002).
DEVELOPMENTAL EXPRESSION PATTERNS OF nAChR α9 AND α10
In adult mammals, each IHC of the organ of Corti transmits the incoming afferent auditory response with contact of up to 20 unbranched afferent fibers (Spoendlin,1972). The OHCs by contrast are largely responsible for mediating the efferent neuronal response through the efferent olivocochlear system. During development prior to the onset of hearing, efferent innervation can be identified on IHCs with demonstration of functional cholinergic synapses, mediated by the nAChR α9 (Uziel et al.,1981; Glowatzki and Fuchs,2000). Additional studies have shown that there is transient α9 expression in immature IHCs as early as embryonic day 18, with the highest levels of expression occurring postnatally at the time of birth in IHCs and near postnatal day 10 in OHCs (Simmons,2002). In IHCs, this expression and subsequent downregulation occurs in a radial pattern, from base to apex. This is followed by a radial pattern of expression of α9 in the OHCs (Zuo et al.,1999).
In contrast to α9 expression occurring primarily prior to synaptogenesis, α10 expression more closely coincides with the arrival of the efferent olivocochlear fibers to the organ of Corti (Simmons,2002). Further α10 has not been identified in adult rat IHCs, while α9 has (Elgoyhen et al.,2001; Morley and Simmons,2002). The α10 subunit also appears to have a later expression pattern than α9, not appearing until E21. Similar to α9, α10 expression appears to be at a maximum in rat IHCs at postnatal day 1 and in OHCs at postnatal day 10. By postnatal day 21, α10 is absent from the IHCs, but remains in the OHCs, a pattern persisting into adulthood (Morley and Simmons,2002). As noted by Simmons (2002), this differential expression pattern suggests synaptic plasticity secondary to medial olivocochlear axon innervation to the organ of Corti. Whole cell electrophysiologic recordings from IHCs in apical turns of the rat organ of Corti add further support to this notion (Katz et al.,2004). These studies show progressively smaller numbers of IHCs with ACh-activated currents between P3 and P14, as compared to P16–22. Further, as the rat ages from P3 through P22, there are progressively smaller numbers of IHCs that demonstrate potassium depolarization of efferent terminals caused inhibitory post-synaptic currents (IPSCs). Taken together, these data suggest that α10, but not α9, is regulated by synaptic activity and support the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits (Katz et al.,2004).
STUDIES IN KNOCKOUT MICE
Important insights into both the function of the nAChR α9α10 as well as the nature of the olivocochlear pathway have been made by studies of nAChR knockout mice. Initial studies of mice lacking a functional nAChR α9 subunit demonstrated that most OHCs were innervated by one large terminal instead of multiple smaller terminals as in wild types, suggesting a role for the nAChR α9 subunit in development of mature synaptic connections. These mice also failed to show suppression of cochlear responses, including compound action potentials and distortion product otoacoustic emissions, during efferent fiber activation, demonstrating the key role α9 receptors play in mediating the only known effects of the olivocochlear system (Vetter et al.,1999). Further behavioral studies on this α9 knockout demonstrated no decrease in tone detection and intensity discrimination in quiet and continuous background noise, as has been shown for mice with olivocochlear pathway lesions, suggesting that central efferent pathways might work in combination with the peripheral olivocochlear system to enhance hearing in noise (May et al.,2002). Additional studies have shown that cochlear sensitivity, based on CAP thresholds, is similar for homozygous mutant and wild-type mice, and that electromotility is also present in OHCs, independent of whether the α9 subunit is present or absent (He et al.,2004). A mouse containing a null mutation for α10 has recently been developed, though there is yet no published data on the functional consequences of this knockout (Vetter et al.,2005).
A transgenic mouse overexpressing the nAChR α9 subunit has also been developed. These mice demonstrate significantly reduced acoustic injury from exposures causing either temporary or permanent damage, without changing preexposure cochlear sensitivity to low- or moderate-level sound (Maison et al.,2002). These data demonstrate that efferent protection is at least in part mediated via the α9 nAChR in the outer hair cells and add further support to the notion that the efferent auditory system, through the olivocochlear pathway, plays an important role in protecting the cochlea from noise injury (Maison and Liberman,2000)
WHAT FOLLOWS ACTIVATION OF OUTER HAIR CELL nAChR α9α10?
As noted above, early studies in isolated chick cochlear hair cells demonstrated a biphasic inhibitory response after the application of ACh (Fuchs and Murrow,1992a,1992b). A multitude of studies have led to a two-channel hypothesis of hair cell inhibition, whereby ACh causes the influx of calcium through the nAChR α9α10, leading to the opening of a small conductance calcium-activated potassium channel, termed the SK2 channel (Fuchs and Murrow,1992b; Martin and Fuchs,1992; Kohler et al.,1996). This highly conserved biphasic response has been seen in all vertebrates studied to date (Art et al.,1984,1985; Blanchet et al.,1996; Evans,1996). Activation of the efferent, biphasic response, either by sound or by direct electrical stimulation, is inhibitory and results in broader tuning and reduced sensitivity of the cochlear afferents (Galambos,1956; Wiederhold and Kiang,1970; Klinke and Galley,1974; Eybalin,1993).
Thus, while the activation of the nAChR α9α10 by the efferent medial olivocochlear fibers is clear, there remain many unanswered questions as to how the SK2 channel becomes activated, and whether or not there may be alternate forms of outer hair cell inhibition mediated through the OHC nAChR. For example, several studies have demonstrated evidence of a G-protein-mediated muscarininc pathway in outer hair cell function. In vivo and in vitro studies in the cochlea have demonstrated that inositol triphosphate (IP3) release can be increased by both carbachol and muscarine at the level of the outer hair cell (Niedzielski and Schacht,1992). Others studies have shown evidence of G-protein-coupled activation of IP3 and calcium following ACh activation of outer hair cells (Shigemoto and Ohmori,1991; Yoshida et al.,1994).
Recent studies also have suggested that OHC inhibition may involve calcium-induced calcium-release mechanisms. Studies by Sridhar et al. (1997) have demonstrated variable mechanisms of calcium activation of the SK channel following nAChR activation, including inhibition over both fast (milliseconds) and slow (seconds) time scales (Sridhar et al.,1995,1997). Despite their different time scales, evidence suggests that the olivocochlear pathway, through nAChR α9α10, mediates both effects (Sridhar et al.,1997; Yoshida et al.,2001). Further, the application of calcium store receptor ryanodine has been shown to alter calcium-induced potassium currents in mammalian OHCs (Evans et al.,2000; Lioudyno et al.,2004), while caffeine, a ryanodine receptor agonist, has been shown to cause shortening and decreased potassium conductance of OHCs (Slepecky et al.,1988; Skellett et al.,1995; Yamamoto et al.,1995).
DOES FUNCTION OF nAChR α9α10 ALSO INVOLVE PROTEIN-PROTEIN INTERACTIONS?
Taken together, these studies strongly suggest that activation of the nAChR α9α10 may have more complicated downstream effects than previously hypothesized. If true, it might be expected that the activation of nAChR α9α10 by acetylcholine could mediate some of these downstream effects directly through protein-protein interactions.
This suggestion is not without precedent since other nAChR subunits have been directly implicated in protein-protein interactions. For example, the intracellular loop of the nAChR subunit α4 has been shown to bind directly to the chaperone protein 14-3-3eta to assist with subunit stabilization (Jeanclos et al.,2001). Additionally, the calcium sensor protein visinin-like protein-1, which is thought to modulate the surface expression and agonist sensitivity of the α4β2 nAChR, has also shown to mediate its interaction through direct binding to the intracellular loop of α4 (Lin et al.,2002). In both these studies, the intracellular loop of the α4 receptor subunit was identified as the site of binding. This is not surprising, since the differences in the amino acid sequence of the cytoplasmic loops largely differentiate the various nAChRs and confer their specificity (Sargent,1993).
To test this hypothesis, a yeast-two-hybrid screen of the nAChR α10 intracellular loop was undertaken against a rat cochlear cDNA library. A genetic screen using an enhanced Gal4 two-hybrid system (Matchmaker System 3; BD Clontech, Palo Alto, CA) was performed as recommended by the manufacturer's protocol, using the intracellular loops of the rat nAChR α10 as bait against a rat cochlear cDNA library. The rat nicotinic acetylcholine receptors α10 (279 bp) intracellular loop was PCR-amplified and subcloned into the Gal 4 DNA binding domain yeast expression vector pGBK T7. The resulting expression vector was transformed into the haploid Y187 competent yeast strain using the lithium acetate method and used as a bait to screen an oligo(dT) cDNA library generated from 0.17 μg polyA RNA purified from 20 cochleas of 3- to 4-week-old rats using TRIzol (Invitrogen, Carlsbad, CA) for total RNA extraction and oligotex mRNA Mini kit (Qiagen, Valencia, CA) to purify mRNA. The cDNAs were PCR-amplified, then purified using a Chroma SPIN-400 column (Clontech) to pool cDNAs of 0.5 Kb or longer and then subcloned into the Gal 4 DNA activating domain pGADT7Rec expression vector before transformation into the haploid yeast strain AH109.
Yeast screening for potential interacting proteins with nAchRs α10 was performed by mating Y187 α10 pGBKT7 and AH109 rat cochlea cDNA library pGADT7-Rec. Diploid or transformants colonies were selected on SD-Ade/-His/-Leu/-Trp+X-α-gal media. Colonies grow in this medium, with a blue colony indicative of a potentially positive interaction between the bait (α10) and the prey (a protein from the library). Potential positive clones activating the reporter genes were restreaked several times on SD-Leu/-Trp+X-α-gal plates, then replica plated on maximally selective SD-Ade/-His/-Leu/-Trp+X-α-gal media ensuring that colonies contained the correct phenotype. The colonies that successfully passed the reporter test were harvested for plasmid isolation using a yeast plasmid isolation kit (USB, Cleveland, OH). AD clones were recovered by selecting on LB agar plates supplemented with ampicillin. Resulting colonies were grown overnight and plasmid DNAs were analyzed by restriction mapping using HaeIII (New England Biolabs) and sequenced to identify the insert in pGADT7Rec vector representing the putative interaction protein. Candidate interacting protein sequences were then Blasted (http://www.ncbi.nlm.nih.gov/BLAST/) for sequence identity.
RT-PCR was then use to test for the presence of mRNA corresponding to the positive clones within the rat cochlea. Total RNA from adult rat inner ear neuroepithelium was isolated using the guanidium-isothiocyanate process (TRIzol RNA Extraction System; Life Technologies) treated with DNAase (1 unit/μg tissue) for 20 min at 37°C and then stored at −20°C.
Purified RNA preparations were subjected to standard reverse transcriptase-polymerase chain reaction (RT-PCR). Using varying amounts of isolated RNA (5 ng to 5 μg) from each tissue source, the RT reaction was allowed to proceed for 60 min at 60°C. PCR was performed for 35 cycles (94°C × 30 sec, 60°C × 45 sec, 72°C × 1 min), followed by agarose gel electrophoresis analysis.
RESULTS AND DISCUSSION
A number of potential binding partners to the α10 subunit intracellular loop were identified, including a protein termed prosaposin. Expression of prosaposin within the cochlea was verified by RT-PCR (Fig. 6). Prosaposin, a precursor of saposins A–D, is a multifunctional protein that has both intra- and extracellular functions, most notably stimulating the hydrolysis of a number of sphingolipids (Kishimoto et al.,1992). Mutations in saposin C have been linked to lysosomal storage disorders such as Gaucher's disease (Horowitz and Zimran,1994). Additionally, prosaposin and saposin C have been shown to act as neurotrophic factors in culture, can bind to a putative G0-coupled cell surface receptor, and may be involved in the prevention of cell death (O'Brien et al.,1994; Hiraiwa et al.,1997; Campana et al.,2000; Misasi et al.,2004). Thus, the identification of prosaposin as a potential binding partner to α10 raises a number of intriguing possibilities as to the nature of nAChR α9α10 function, including additional G-protein-related muscarinic-like properties of the receptor.
The outer hair cell nAChR α9α10 is a unique receptor. It has a characteristic mixed nicotinic-muscarinic pharmacologic profile and is sensitive to a broad range of agonists and antagonists. Though nearly always identified together in adult tissues, there does appear to be differing regulatory mechanisms of each subunit of the functional receptor. Studies on the nAChR α9α10 have greatly enhanced our understanding of nAChR function, the efferent olivocochlear pathway, and auditory hair cell function and development. Further, the nAChR α9α10 clearly has a function outside of the auditory system, though this precise function remains to be fully elucidated. As studies further define the roles of this important receptor in physiology, it may 1 day represent an important component of human disease and a potential target for therapy for a variety of ear disorders, including preventing or treating noise-induced hearing loss, or such debilitating disorders as vertigo or tinnitus. The neuronal AChR superfamily has now been implicated in several diseases. In both Alzheimer's disease and Parkinson's disease, the number of nicotine binding sites (presumably α4β2 AChRs) is decreased (Whitehouse et al.,1988). Mutated forms of nAChRs have also been shown to underlie many forms of congenital myasthenic syndromes, including slow-channel syndrome myasthenia and low-affinity fast-channel syndrome myasthenia (Ohno et al.,1993,1996; Engel et al.,1996; Croxen et al.,1998; Singh et al.,2004). Additional disorders attributable to nAChRs include nocturnal frontal lobe epilepsy (Steinlein et al.,1995) and schizophrenia (Vafa and Schofield,1998). It is thus likely that the nAChR α9α10 may also be implicated as an agent of disease within the inner ear.