Cloning and characterization of α9 subunits of the nicotinic acetylcholine receptor expressed by saccular hair cells of the rainbow trout (Oncorhynchus mykiss)

Drescher, Dennis G.; Ramakrishnan, Neeliyath A.; Drescher, Marian J.; Chun, Won-Kyong; Wang, X; Myers, Steven F.; Green, G.E; Sadrazodi, K; Karadaghy, Ahmad A; Poopat, N; Karpenko, A.N; Khan, Khalid M.; Hatfield, James S.

doi:10.1016/j.neuroscience.2004.05.037

Cited by 23 publications

(18 citation statements)

References 54 publications

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“…Moreover, it has been described that this fish genome contains the largest family of vertebrate nAChR subunits reported to date (52). In addition three ␣9 subunit genes have been described in the rainbow trout (Oncorhynchus mykiss) (53). Although none of the Fugu nAChR sequences show close identity to the mammalian or avian ␣10 subunits, three of the ␣9-like subunits possess glycosylation sites also found in the higher vertebrate ␣10 subunit (52), suggesting that these ␣9-like subunits might coassemble to form a functional nAChR, much like the higher vertebrate homomeric ␣9 and/or heteromeric ␣9␣10 nAChRs.…”

Section: Discussionmentioning

confidence: 99%

A Novel α-Conotoxin, PeIA, Cloned from Conus pergrandis, Discriminates between Rat α9α10 and α7 Nicotinic Cholinergic Receptors

McIntosh

Plazas

Watkins

et al. 2005

Journal of Biological Chemistry

121

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The ␣9 and ␣10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of ␣9 and ␣10 overlaps with that of ␣7 in the cochlea and in sites such as dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes, and skin. We now report the cloning, total synthesis, and characterization of a novel toxin ␣-conotoxin PeIA that discriminates between ␣9␣10 and ␣7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. ␣-Conotoxin PeIA displayed a 260-fold higher selectivity for ␣-bungarotoxin-sensitive ␣9␣10 nAChRs compared with ␣-bungarotoxin-sensitive ␣7 receptors. The IC 50 of the toxin was 6.9 ؎ 0.5 nM and 4.4 ؎ 0.5 nM for recombinant ␣9␣10 and wild-type hair cell nAChRs, respectively. ␣-Conotoxin PeIA bears high resemblance to ␣-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither ␣-conotoxin MII nor ␣-conotoxin GIC at concentrations of 10 M blocked acetylcholine responses elicited in Xenopus oocytes injected with the ␣9 and ␣10 subunits. Among neuronal non-␣-bungarotoxin-sensitive receptors, ␣-conotoxin PeIA was also active at ␣3␤2 receptors and chimeric ␣6/␣3␤2␤3 receptors. ␣-Conotoxin PeIA represents a novel probe to differentiate responses mediated either through ␣9␣10 or ␣7 nAChRs in those tissues where both receptors are expressed.

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Section: Discussionmentioning

confidence: 99%

A Novel α-Conotoxin, PeIA, Cloned from Conus pergrandis, Discriminates between Rat α9α10 and α7 Nicotinic Cholinergic Receptors

McIntosh

Plazas

Watkins

et al. 2005

Journal of Biological Chemistry

121

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“…Immunoreactivity was concentrated at apical, supranuclear sites of hair cells, extending to the lateral and basal cell membrane (Fig. 4B), which are sites of efferent input (17). In addition, HCN1 was immunolocalized to stereocilia, with discrete sites of expression that were gradated in their distribution (Fig.…”

Section: Immunolocalization Of Hcn1 In Hair Cells Of the Trout Sacculmentioning

confidence: 95%

Calcium-dependent Binding of HCN1 Channel Protein to Hair Cell Stereociliary Tip Link Protein Protocadherin 15 CD3

Ramakrishnan

Drescher

Barretto

et al. 2009

Journal of Biological Chemistry

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The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform expressed in trout saccular hair cells, was found by yeast two-hybrid protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin 15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair cell stereocilia, consistent with gradated distribution expected for tip link sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries of rat cochlear inner and outer hair cells, and HCN1 protein was immunolocalized to cochlear hair cell stereocilia. As predicted by the trout hair cell model, the amino terminus of rat organ of Corti HCN1 was found by yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15 CD3, a tip link protein implicated in mechanosensory transduction. Specific binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down assays and surface plasmon resonance analysis, both predicting dependence on Ca HCN12 is the primary full-length HCN isoform underlying I h (hyperpolarization-activated, cyclic nucleotide-gated, nonselective cation channel current) in a model hair cell preparation from the trout sacccule (1). cAMP-gated I h , possibly in addition to the mechanosensory-transduction current, sets the membrane potential for a subpopulation of saccular hair cells (2, 3). The membrane potential in the saccular hair cell subpopulation is sufficiently depolarized to activate voltage-gated calcium channels, permitting influx of calcium and secretion of hair cell transmitter (2). Given that saccular hair cells expressing I K1 in addition to I h are more hyperpolarized, not supporting activation of the voltage-gated calcium channels, we predicted that spontaneous release of transmitter from the subpopulation of hair cells would constitute hair cell-generated spontaneous activity for the saccule (1). However, little has been previously reported on the morphological localization of the HCN1 isoform in hair cells or possible links to structural proteins that mechanistically would localize HCN1 in hair cells (for preliminary report, see Ref. 4). In general, little is known about proteinprotein interactions for the HCN isoforms that would modulate I h and/or the associated instantaneous current (5).Protocadherin 15 is a proposed tip link protein involved in connecting shorter stereocilia to adjacent taller stereocilia in the stereociliary array of inner ear hair cells, facilitating the opening of the mechanosensory transduction channel in response to auditory and vestibular stimuli. The active tip link protein in Danio rerio is protocadherin 15a (6), characterized by splice variants in its carboxyl terminus. In the mammal, protocadherin 15 CD3 is hypothesized to be a tip link protein at insertion sites in the tips of the shorter stereocilia of the stereociliary array (7,8). EXPERIMENTAL PROCEDURES Acquisition of a Model Hair Cell Preparation from the Trout Saccule and an Organ of Corti Subfraction from theRat Cochlea-Hair cell layers were isolated from the t...

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“…The question of AC isoform expression and Gαs/olf subunit expression in vestibular hair cells has now been directly addressed for a model hair cell preparation from the trout saccule for which the hair cell is the only intact cell type, a primary source of full-length sequence of proteins and splice variants expressed in hair cells (Ramakrishnan et al, 2002; Cho et al, 2003; Drescher DG et al, 2004; Ramakrishnan et al, 2006). With this hair cell model preparation, we have obtained evidence for an adenylyl cyclase pathway comprising the specific AC isoforms and G protein α subunits that are utilized in dopamine receptor signal transduction (Herve et al, 1993; Zhuang et al, 2000; Iwamoto et al, 2003; Lee et al, 2002), pointing to the possibility of dopamine receptor expression by vestibular hair cells.…”

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An adenylyl cyclase signaling pathway predicts direct dopaminergic input to vestibular hair cells

et al. 2010

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Adenylyl cyclase signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across adenylyl cyclase (AC) isoforms, and RT-PCR, coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca2+, the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca2+. Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate adenylyl cyclase activation in the absence of Gαs in mammalian cochlear hair cells, the issue of hair-cell Gαs mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for Gαs/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for Gαi have also been detected in the hair cell layer, one with homology to Gαi1 and the second with homology to Gαi3 of higher vertebrates. Both Gαs/olf protein and Gαi1/Gαi3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. While a signaling event coupling to Gαs/olf and Gαi1/Gαi3 in the stereocilia is currently unknown, signaling with Gαs/olf, Gαi3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to Gαolf and activation of AC5/6. Co-expression with dopamine D2 receptor, which itself couples to Gαi3 and AC5/6, will down-modulate levels of cAMP, thus fine-tuning and gradating the hair-cell response to dopamine D1A. As predicted by the trout saccular hair cell model, evidence has been obtained for the first time that hair cells of mammalian otolithic vestibular end organs (rat/mouse saccule/utricle) express dopamine D1A and D2L receptors, and each receptor co-localizes with AC5/6, with a marked presence of all three proteins in subcuticular regions of type I vestibular hair cells. A putative efferent, presynaptic source of dopamine was identified in tyrosine hydroxylase-positive nerve fibers which passed from underlying connective tissue to the sensory epithelia, ending on type I and type II vestibular hair cells and on afferent calyces.

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Cloning and characterization of α9 subunits of the nicotinic acetylcholine receptor expressed by saccular hair cells of the rainbow trout (Oncorhynchus mykiss)

Cited by 23 publications

References 54 publications

A Novel α-Conotoxin, PeIA, Cloned from Conus pergrandis, Discriminates between Rat α9α10 and α7 Nicotinic Cholinergic Receptors

A Novel α-Conotoxin, PeIA, Cloned from Conus pergrandis, Discriminates between Rat α9α10 and α7 Nicotinic Cholinergic Receptors

Calcium-dependent Binding of HCN1 Channel Protein to Hair Cell Stereociliary Tip Link Protein Protocadherin 15 CD3

An adenylyl cyclase signaling pathway predicts direct dopaminergic input to vestibular hair cells

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