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Cellular localization of voltage-gated calcium channels and synaptic vesicle-associated proteins in the guinea pig cochlea

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Abstract

The cellular localization of voltage-gated calcium channels (VGCCs) and synaptic vesicle-associated proteins, SV2, synapsin I, and vesicle-associated membrane protein (VAMP) (synaptobrevin), was investigated in the guinea pig cochlea using immunocytochemistry and confocal laser scanning microscopy. Reactivity, in guinea pig, of antibodies to the α1 subunits of L-type, α1C [Cav1.2] and α1D [Cav1.3]; P/Q-type, α1A [Cav2.1]; and R-type, α1E [Cav2.3] high voltage-activated calcium channels, was determined by Western blotting and immunolabeling of cerebellum. In the cochlea the sensory inner hair cells of the organ of Corti displayed strong intracellular staining, predominantly localized to their basolateral poles, with an antibody directed against the α1C subunit. Some α1C labeling was also observed in the inner pillar cells, in cell bodies of afferent neurons in the spiral ganglion, and in the inferior region of the spiral ligament. The supporting pillar cells were strongly immunoreactive throughout for α1D, but no α1D labeling of the inner hair cells was seen. The α1A subunit showed a cytoplasmic distribution in all three rows of outer hair cells. α1E labeling localized to the outer hair cells, predominantly in the subcuticular plate region, and also to nerve fiber bundles beneath these hair cells. Strong immunoreactivity was consistently seen with antibodies directed against SV2 and synapsin I in neuronal structures surrounding the basolateral surfaces of both the inner and outer hair cells but was absent from the sensory cells themselves. VAMP labeling was found throughout the cytoplasm of the inner hair cells and in neuronal structures beneath the hair cells. These results reveal a differential distribution of VGCC-types in the sensory and nonsensory elements of the guinea pig cochlea, with the inner hair cells expressing α1C L-type channels and VAMP but not synapsin I or SV2.

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References

  • Atlas D. (2001) Functional and physical coupling of voltage-sensitive calcium channels with exocytotic proteins: ramifications for the secretion mechanism. J. Neurochem. 77, 972–985.

    Article  PubMed  CAS  Google Scholar 

  • Beutner D., Voets T., Neher E., and Moser T. (2001) Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse. Neuron 29, 681–690.

    Article  PubMed  CAS  Google Scholar 

  • Bobbin R. P., Jastreboff P. J., Fallon M., and Littman T. (1990) Nimodipine, an L-channel Ca2+ antagonist, reverses the negative summating potential recorded from the guinea pig cochlea. Hear. Res. 46, 277–287.

    Article  PubMed  CAS  Google Scholar 

  • Bolt M. W. and Mahoney P. A. (1997) High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 247, 185–192.

    Article  PubMed  CAS  Google Scholar 

  • Brandt A., Striessnig J., and Moser T. (2003) CaV1.3 channels are essential for development and presynaptic activity of cochlear inner hair cells. J. Neurosci. 23, 10832–10840.

    PubMed  CAS  Google Scholar 

  • Brice N. L. and Dolphin A. C. (1999) Differential plasma membrane targeting of voltage-dependent calcium channel subunits expressed in a polarized epithelial cell line. J. Physiol. 515, 685–694.

    Article  PubMed  CAS  Google Scholar 

  • Chung J. W. and Schacht J. (2001) ATP and nitric oxide modulate intracellular calcium in isolated pillar cells of the guinea pig cochlea. JARO 2, 399–407.

    Article  PubMed  CAS  Google Scholar 

  • Erickson M. G., Liang H., Mori M. X., and Yue D. T. (2003) FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation. Neuron 39, 97–107

    Article  PubMed  CAS  Google Scholar 

  • Ertel E. A., Campbell K. P., Harpold M. M., Hofmann F., Mori Y., Perez-Reyes E., et al. (2000) Nomenclature of voltage-gated calcium channels. Neuron 25, 533–535.

    Article  PubMed  CAS  Google Scholar 

  • Feany M. B., Lee S., Edwards R. H., and Buckley K. M. (1992) The synaptic vesicle protein SV2 is a novel type of transmembrane transporter. Cell 70, 861–867.

    Article  PubMed  CAS  Google Scholar 

  • Fettiplace R. and Fuchs P. A. (1999) Mechanisms of hair cell tuning. Annu. Rev. Physiol. 61, 809–834.

    Article  PubMed  CAS  Google Scholar 

  • Fisher T. E. and Bourque C. W. (2001) The function of Ca2+ channel subtypes in exocytotic secretion: new perspectives from synaptic and non-synaptic release. Prog. Biophys. Mol. Biol. 77, 269–303.

    Article  PubMed  CAS  Google Scholar 

  • Fisher T. E., Carrion-Vazquez M., and Fernandez J. M. (2000) Intracellular Ca2+ channel immunoreactivity in neuroendocrine axon terminals. FEBS Lett. 482, 131–138.

    Article  PubMed  CAS  Google Scholar 

  • Flock Å, Flock B, Fridberger A, Scarfone E, and Ulfendahl M. (1999) Supporting cells contribute to control of hearing sensitivity. J. Neurosci. 19, 4498–4507.

    PubMed  CAS  Google Scholar 

  • Glowatzki E. and Fuchs P. A. (2002) Transmitter release at the hair cell ribbon synapse. Nat. Neurosci. 5, 147–154.

    Article  PubMed  CAS  Google Scholar 

  • Green G. E., Khan K. M., Beisel K. W., Drescher M. J., Hatfield J. S., and Drescher D. G. (1996) Calcium channel subunits in the mouse cochlea. J. Neurochem. 67, 37–45.

    Article  Google Scholar 

  • Hell J. W., Westenbroek R. E., Warner C., Ahlijanian M. K., Prystay W., Gilbert M. M., et al. (1993) Identification and differential subcellular localization of the neuronal class C and class DL-type calcium channel α1 subunits. J. Cell. Biol. 123, 949–962.

    Article  PubMed  CAS  Google Scholar 

  • Hofmann F., Lcinova L., and Klugbauer N. (1999) Voltage-dependent calcium channels: from structure to function. Rev. Physiol. Biochem. Pharmacol. 139, 33–87.

    Article  PubMed  CAS  Google Scholar 

  • Housley G. D. and Ashmore J. F (1992) Ionic currents of outer hair cells isolated from the guinea pig cochlea. J. Physiol. 448, 73–98.

    PubMed  CAS  Google Scholar 

  • Hu K., Carroll J., Fedorovich S., Rickman C., Sukhodub A, and Davletov B. (2002) Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion. Nature 415, 646–650.

    Article  PubMed  CAS  Google Scholar 

  • Hu X.-Q., Singh N., Mukhopadhyay D., and Akbarali H. I. (1998) Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion kinase. J. Biol. Chem. 273, 5337–5342.

    Article  PubMed  CAS  Google Scholar 

  • Hui A., Ellinor P. T., Krizanova O., Wang J.-J., Diebold R. J., and Schwartz A. (1991) Molecular cloning of multiple subtypes of a novel rat brain isoform of the α1 subunit of the voltage-dependent channel. Neuron 7, 35–44.

    Article  PubMed  CAS  Google Scholar 

  • Ikeda S. R. (2001) Calcium channels—link locally, act globally. Science 294, 318–319.

    Article  PubMed  CAS  Google Scholar 

  • Issa N. P. and Hudspeth A. J. (1996) The entry and clearance of Ca2+ at individual presynaptic active zones of hair cells from the bullfrog’s sacculus. Proc. Natl. Acad. Sci. U. S. A. 93, 9527–9532.

    Article  Google Scholar 

  • Kollmar R., Montgomery L. G., Fak J., Henry L J., and Hudspeth A. J. (1997) Predominance of the α1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken’s cochlea. Proc. Natl. Acad. Sci. U. S. A. 94, 14883–14888.

    Article  PubMed  CAS  Google Scholar 

  • Kreuzberg U., Theissen P., Schicha H., Schröder F., Mehlhorn U., De Vivie E. R., et al. (2000) Single-channel activity and expression of atrial L-type Ca2+ channels in patients with latent hyperthyroidism. Am. J. Physiol. Heart Circ. Physiol. 278, H723-H730.

    PubMed  CAS  Google Scholar 

  • Lelli A., Perin P., Martini M., Ciubotaru C. D., Prigioni I., Valli P., et al. (2003) Presynaptic calcium stores modulate afferent release in vestibular hair cells. J. Neurosci. 23, 6894–6903.

    PubMed  CAS  Google Scholar 

  • Lenzi D. and von Gersdorff H. (2001) Structure suggests function: the case for synaptic ribbons as exocytotic nanomachines. BioEssays 23, 831–840.

    Article  PubMed  CAS  Google Scholar 

  • Liang F., Hu W., Schulte B. A., Mao C, Qu C, Hazen-Martin D. J., and Shen Z. (2004) Identification and characterization of an L-type Cav1.2 channel in spiral ligament fibrocytes of the gerbil inner ear. Mol. Brain Res. 125, 40–46.

    Article  PubMed  CAS  Google Scholar 

  • Liberman M. C. and Oliver M. E. (1984) Morphometry of intracellularly labeled neurons of the auditory nerve: correlations with functional properties. J. Comp. Neurol. 223, 163–176.

    Article  PubMed  CAS  Google Scholar 

  • Lopez I., Ishiyama G., Acuna D., Ishiyama A., and Baloh R. W. (2003) Immunolocalization of voltage-gated calcium channel α1 subunits in the chinchilla cochlea. Cell Tissue Res. 313, 177–186.

    Article  PubMed  CAS  Google Scholar 

  • Lopez I., Ishiyama G., Ishiyama A., Jen J. C., Liu F., and Baloh R. W. (1999) Differential subcellular immunolocalization of voltage-gated calcium channel α1 subunits in the chinchilla cristae ampullaris. Neuroscience 92, 773–782.

    Article  PubMed  CAS  Google Scholar 

  • Mammano F., Frolenkov G. I., Lagostena L., Belyantseva I. A., Kurc M., Dodane V., et al. (1999) ATP-induced Ca2+ release in cochlear outer hair cells: localization of an inositol triphosphate-gated Ca2+ store to the base of the sensory hair bundle. J. Neurosci. 19, 6918–6929.

    PubMed  CAS  Google Scholar 

  • McLean I. W. and Nakane P. K. (1974) Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077–1083.

    PubMed  CAS  Google Scholar 

  • Merchan-Perez A. and Liberman M. C. (1996) Ultrastructural differences among afferent synapses on cochlear hair cells: correlations with spontaneous discharge rate. J. Comp. Neurol. 371, 208–221.

    Article  Google Scholar 

  • Mese G., Londin E., Mui R., Brink P. R., and White T. W. (2004) Altered gating properties of functional Cx26 mutants associated with recessive non-syndromic hearing loss. Hum. Genet. 115, 191–199.

    Article  PubMed  CAS  Google Scholar 

  • Muller M. and Robertson D. (1991a) Relationship between tone burst discharge pattern and spontaneous firing rate of auditory nerve fibres in the guinea pig. Hear. Res. 57, 63–70.

    Article  PubMed  CAS  Google Scholar 

  • Muller M. and Robertson D. (1991b) Shapes of rate-versus-level functions of primary auditory nerve fibres: test of the basilar membrane mechanical hypothesis. Hear. Res. 57, 71–78.

    Article  PubMed  CAS  Google Scholar 

  • Nachman-Clewner M., St. Jules R., and Townes-Anderson E. (1999) L-type calcium channels in the photoreceptor ribbon synapse: localization and role in plasticity. J. Comp. Neurol. 415, 1–16.

    Article  PubMed  CAS  Google Scholar 

  • Obermair G. J., Szabo Z., Bourinet E., and Flucher B. E. (2004) Differential targeting of the L-type Ca2+ channel α1C (Cav1.2) to synaptic and extrasynaptic compartments in hippocampal neurons. Eur. J. Neurosci. 19, 2109–2122.

    Article  PubMed  Google Scholar 

  • Oshima T., Ikeda K., Furukawa M., Ueda N., Suzuki H., and Takasaka T. (1996) Distribution of Ca2+ channels on cochlear outer hair cells revealed by fluorescent dihydropyridines. Am. J. Physiol. 271 (Cell Physiol. 40), C944-C949.

    Google Scholar 

  • Patuzzi R. and Robertson D. (1988) Tuning in the mammalian cochlea. Physiol. Rev. 68, 1009–1082.

    PubMed  CAS  Google Scholar 

  • Platzer J., Engel J., Schrott-Fischer A., Stephan K., Bova S., Chen H., et al. (2000) Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+ channels. Cell 102, 89–97.

    Article  PubMed  CAS  Google Scholar 

  • Raphael Y. and Altschuler R. A. (2003) Structure and innervation of the cochlea. Brain Res. Bull. 60, 397–422.

    Article  PubMed  Google Scholar 

  • Roberts W. M., Jacobs R. A., and Hudspeth A. J. (1990) Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. J. Neurosci. 10, 3664–3684.

    PubMed  CAS  Google Scholar 

  • Robertson D. and Johnstone B. M. (1979) Effect of divalent cations on spontaneous and evoked activity of single mammalian auditory neurones. Pflügers Arch. 380, 7–12.

    Article  PubMed  CAS  Google Scholar 

  • Robertson D. and Paki B. (2002) Role of L-type Ca2+ channels in transmitter release from mammalian inner hair cells. II. Single-neuron activity. J. Neurophysiol. 87, 2734–2740.

    PubMed  CAS  Google Scholar 

  • Robertson D., Sellick P. M., and Patuzzi R. (1999) The continuing search for outer hair cell afferents in the guinea pig spiral ganglion. Hear. Res. 136, 151–158.

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez-Contreras A. and Yamoah E. N. (2001) Direct measurement of single-channel Ca2+ currents in bull-frog hair cells reveals two distinct channel subtypes. J. Physiol. 534, 669–689.

    Article  PubMed  CAS  Google Scholar 

  • Rosahl T. W., Spillane D., Missler M., Herz J., Selig D. K., Wolff J. R., et al. (1995) Essential functions of synapsins I and II in synaptic vesicle regulation. Nature 375, 488–493.

    Article  PubMed  CAS  Google Scholar 

  • Saegusa H., Kurihara T., Zang S., Minowa O., Kazuno A., Han W., et al. (2000) Altered pain responses in mice lacking α1E subunit of the voltage-dependent Ca2+ channel. Proc. Natl. Acad. Sci. U. S. A. 97, 6132–6137.

    Article  PubMed  CAS  Google Scholar 

  • Safieddine S. and Wenthold R. J. (1999) SNARE complex at the ribbon synapses of cochlear hair cells: analysis of synaptic vesicle-and synaptic membrane-associated proteins. Eur. J. Neurosci. 11, 803–812.

    Article  PubMed  CAS  Google Scholar 

  • Schneggenburger R. and Neher E. (2000) Intracellular calcium dependence of transmitter release rates at a fast central synapse. Nature 406, 889–893.

    Article  PubMed  CAS  Google Scholar 

  • Schramm M., Vajna R., Pereverzev A., Tottene A., Klöckner U., Pietrobon D., et al. (1999) Isoforms of α1E voltage-gated calcium channels in rat cerebellar granule cells—detection of major calcium channel α1-transcripts by reverse transcription-polymerase chain reaction. Neuroscience 92, 565–575.

    Article  PubMed  CAS  Google Scholar 

  • Sheng Z. H., Westenbroek R. E., and Catterall W. A. (1998) Physical link and functional coupling of presynaptic calcium channels and the synaptic vesicle docking/fusion machinery. J. Bioenerg. Biomembr. 30, 335–345.

    Article  PubMed  CAS  Google Scholar 

  • Slepecky N. B. (1996) Structure of the mammalian cochlea, in The Cochlea, Dallos, P., Popper, A. N., and Fay, R. R., eds., Springer, New York, pp. 44–129.

    Google Scholar 

  • Slepecky N. B. and Ulfendhal M. (1993) Evidence for calcium-binding proteins and calcium-dependent regulatory proteins in sensory cells of the organ of Corti. Hear. Res. 70, 73–84.

    Article  PubMed  CAS  Google Scholar 

  • Snutch T. P., Tomlinson J. W., Leonard J. P., and Gilbert M. M. (1991) Distinct calcium channels are generated by alternative splicing and are differentially expressed in the mammalian CNS. Neuron 7, 45–57.

    Article  PubMed  CAS  Google Scholar 

  • Sobkowicz H. M., August B. K., and Slapnick S. M. (2004) Synaptic arrangements between inner hair cells and tunnel fibers in the mouse cochlea. Synapse 52, 299–315.

    Article  PubMed  CAS  Google Scholar 

  • Steel K. P. and Kros C. J. (2001) A genetic approach to understanding auditory function. Nat. Genet. 27, 143–149.

    Article  PubMed  CAS  Google Scholar 

  • Sueta T., Zhang S. Y., Sellick P. M., Patuzzi R., and Robertson D. (2004) Effects of a calcium channel blocker on spontaneous neural noise and gross action potential waveforms in the guinea pig cochlea. Hear. Res. 188, 117–125.

    Article  PubMed  CAS  Google Scholar 

  • Tsuji J. and Liberman M. C. (1997) Intracellular labeling of auditory nerve fibers in guinea pig: central and peripheral projections. J. Comp. Neurol. 381, 188–202.

    Article  PubMed  CAS  Google Scholar 

  • Tucker T. and Fettiplace R. (1995) Confocal imaging of calcium microdomains and calcium extrusion in turtle hair cells. Neuron 15, 1323–1335.

    Article  PubMed  CAS  Google Scholar 

  • Waka N., Knipper M., and Engel J. (2003) Localization of the calcium channel subunits Cav1.2 (alpha1C) and Cav2.3 (alpha1E) in the mouse organ of Corti. Histol. Histopathol. 18, 1115–1123.

    PubMed  CAS  Google Scholar 

  • Westenbroek R. E., Sakurai T., Elliott E. M., Hell J. W., Starr T. V. B., Snutch T. P., and Catterall W. A. (1995) Immunochemical identification and subcellular distribution of the α1A subunits of brain calcium channels. J. Neurosci. 15, 6403–6418.

    PubMed  CAS  Google Scholar 

  • Winter I. M., Robertson D., and Yates G. K. (1990) Diversity of characteristic frequency rate-intensity functions in guinea pig auditory nerve fibres. Hear. Res. 45, 191–202.

    Article  PubMed  CAS  Google Scholar 

  • Wiser O., Trus M., Hernández A., Renström E., Barg S., Rorsman P., and Atlas D. (1999) The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery. Proc. Natl. Acad. Sci. U. S. A. 96, 248–253.

    Article  PubMed  CAS  Google Scholar 

  • Xu T. and Bajjalieh S. M. (2001) SV2 modulates the size of the readily releasable pool secretory vesicles. Nat. Cell Biol. 3, 691–698.

    Article  PubMed  CAS  Google Scholar 

  • Yang S.-N., Larsson O., Bränstörm R., Bertorello A. M., Leibiger B., Leibiger I. B., et al. (1999) Syntaxin 1 interacts with the LD subtype of voltage-gated Ca2+ channels in pancreatic β cells. Proc. Natl. Acad. Sci. U. S. A. 96, 10164–10169.

    Article  PubMed  CAS  Google Scholar 

  • Yokoyama C. T., Westenbroek R. E., Hell J. W., Soong T. W., Snutch T. P., and Catterall W. A. (1995) Biochemical properties and subcellular distribution of the neuronal class E calcium channel alpha 1 subunit. J. Neurosci. 15, 6419–6432.

    PubMed  CAS  Google Scholar 

  • Zenisek D., Davila V., Wan L., and Almers W. (2003) Imaging calcium entry sites and ribbon structures in two presynaptic cells. J. Neurosci. 23, 2538–2548.

    PubMed  CAS  Google Scholar 

  • Zhang J.-F., Randall A. D., Ellinor P. T., Horne W. A., Sather W. A., Tanabe T., et al. (1993) Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons. Neuropharmacology 32, 1075–1088.

    Article  PubMed  CAS  Google Scholar 

  • Zhang S. Y., Robertson D., Yates G., and Everett A. (1999) Role of L-type Ca2+ channels in transmitter release from mammalian inner hair cells 1. Gross sound-evoked potentials. J. Neurophysiol. 82, 3307–3315.

    PubMed  CAS  Google Scholar 

  • Zhao H. B. and Santos-Sacchi J. (2000) Voltage gating of gap junctions in cochlear supporting cells: evidence for nonhomotypic channels. J. Membr. Biol. 175, 17–24.

    Article  PubMed  CAS  Google Scholar 

  • Zhou H., Kim S.-A., Kirk E. A., Tippens A. L., Sun H., Haeseleer F., and Lee A. (2004) Ca2+- binding protein facilitates and forms a postsynaptic complex with Cav1.2 (L-type)Ca2−channels. J. Neurosci. 24, 4698–4708.

    Article  PubMed  CAS  Google Scholar 

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  1. The Auditory Laboratory, Synapse Laboratory, Biomolecular and Chemical Sciences, The University of Western Australia, 6009, Crawley, Western Australia, Australia

    Maria G. Layton, Donald Robertson, Wilhelmina H. A. M. Mulders & Graeme K. Yates

  2. Auditory Laboratory Physiology, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 6009, Crawley, Western Australia, Australia

    Alan W. Everett

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  1. Maria G. Layton
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  2. Donald Robertson
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Layton, M.G., Robertson, D., Everett, A.W. et al. Cellular localization of voltage-gated calcium channels and synaptic vesicle-associated proteins in the guinea pig cochlea. J Mol Neurosci 27, 225–244 (2005). https://doi.org/10.1385/JMN:27:2:225

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