Expression of adenylyl cyclase type I in cochlear inner hair cells

Drescher, Marian J.; Khan, Khalid M.; Beisel, Kirk W.; Karadaghy, Ahmad A; Hatfield, James S.; Kim, Steve Y; Drescher, Andrew J.; Lasak, John M; Barretto, Roberto L.; Shakir, Ali; Drescher, Dennis G.

doi:10.1016/s0169-328x(97)00007-7

Cited by 22 publications

(8 citation statements)

References 22 publications

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“…AC7 activity is independent of Ca 2+ . These results for a type II-like vestibular hair cell differ from cochlear inner hair cell expression of AC isoforms (Drescher et al, 1997), consistent with the hypothesis that hair cells of different types, cochlear inner and outer hair cells, and type I and type II vestibular hair cells, differ in AC-modulated function.…”

Section: Discussionsupporting

confidence: 85%

“…The identification of calmodulin as a hair cell marker localized to the stereocilia (Slepecky et al, 1988; Shepherd et al, 1989; Slepecky and Ulfendahl, 1993; Ogata and Slepecky, 1998) suggested an alternative mechanism to activate AC isoforms in the absence of Gαs. Message and protein for ACI, one of the Ca 2+ /calmodulin-activated AC isoforms, was subsequently localized to cochlear inner hair cells (Drescher et al, 1997). Little information on adenylyl cyclase signal transduction pathways in vestibular hair cells has been available.…”

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confidence: 99%

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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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Section: Discussionsupporting

confidence: 85%

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confidence: 99%

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

et al. 2010

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“…After ligand recognition, class B GPCRs activate G-proteins containing G␣s subunits that stimulate ACs, hence cAMP production. AC1, AC4, AC6, AC7, and AC9 transcripts have been found in the mouse and rat organs of Corti by RT-PCR analysis (Drescher et al, 1997;Kumagami et al, 1999). We studied the expression of AC isoforms in the mouse cochlear hair cells by single-cell RT-PCR analysis, as described previously (Adato et al, 2005b).…”

Section: Lack Of Vlgr1 Affects the Stereociliar Distribution Of Adenymentioning

confidence: 99%

Molecular Characterization of the Ankle-Link Complex in Cochlear Hair Cells and Its Role in the Hair Bundle Functioning

Michalski¹,

Michel²,

Bahloul³

et al. 2007

Journal of Neuroscience

185

283

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Several lines of evidence indicate that very large G-protein-coupled receptor 1 (Vlgr1) makes up the ankle links that connect the stereocilia of hair cells at their base. Here, we show that the transmembrane protein usherin, the putative transmembrane protein vezatin, and the PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-containing submembrane protein whirlin are colocalized with Vlgr1 at the stereocilia base in developing cochlear hair cells and are absent in Vlgr1 Ϫ/Ϫ mice that lack the ankle links. Direct in vitro interactions between these four proteins further support their involvement in a molecular complex associated with the ankle links and scaffolded by whirlin. In addition, the delocalization of these proteins in myosin VIIa defective mutant mice as well as the myosin VIIa tail direct interactions with vezatin, whirlin, and, we show, Vlgr1 and usherin, suggest that myosin VIIa conveys proteins of the ankle-link complex to the stereocilia. Adenylyl cyclase 6, which was found at the base of stereocilia, was both overexpressed and mislocated in Vlgr1 Ϫ/Ϫ mice. In postnatal day 7 Vlgr1 Ϫ/Ϫ mice, mechanoelectrical transduction currents evoked by displacements of the hair bundle toward the tallest stereocilia (i.e., in the excitatory direction) were reduced in outer but not inner hair cells. In both cell types, stimulation of the hair bundle in the opposite direction paradoxically resulted in significant transduction currents. The absence of ankle-linkmediated cohesive forces within hair bundles lacking Vlgr1 may account for the electrophysiological results. However, because some long cadherin-23 isoforms could no longer be detected in Vlgr1 Ϫ/Ϫ mice shortly after birth, the loss of some apical links could be involved too. The premature disappearance of these cadherin isoforms in the Vlgr1 Ϫ/Ϫ mutant argues in favor of a signaling function of the ankle links in hair bundle differentiation.

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“…Primers for PCR targeting rat HCN1 (GenBank TM accession number NM_053375) were 5Ј-CAGCAGCCGCAA-CAGCAACAA-3Ј upstream (nucleotides 2328 -2348), and 5Ј-TTTTCTGCATCTGGGTCTTTATTTAAGA-3Ј downstream (nucleotides 2755-2782), predicted to yield a 455-bp amplification product. The expression of HCN1 message in cochlear hair cells was determined from application of ratspecific HCN1 primers in PCR to ZAP cDNA libraries (a cDNA library formed with a specifically modified phage vector, ZAP (Stratagene)) (14,15). A first amplification with an HCN1 gene-specific primer upstream coupled with ZAP-T7 reverse primer downstream was followed by a second amplification with HCN1 gene-specific primers for the 455-bp PCR product.…”

Section: Acquisition Of a Model Hair Cell Preparation From The Trout mentioning

confidence: 99%

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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Expression of adenylyl cyclase type I in cochlear inner hair cells

Cited by 22 publications

References 22 publications

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

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

Molecular Characterization of the Ankle-Link Complex in Cochlear Hair Cells and Its Role in the Hair Bundle Functioning

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

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