Cargando…

Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells

Efferent cholinergic neurons inhibit sensory hair cells of the vertebrate inner ear through the combined action of calcium-permeable α9α10-containing nicotinic acetylcholine receptors (nAChRs) and associated calcium-dependent potassium channels. The venom of cone snails is a rich repository of bioac...

Descripción completa

Detalles Bibliográficos
Autores principales: Fisher, Fernando, Zhang, Yuanyuan, Vincent, Philippe F. Y., Gajewiak, Joanna, Gordon, Thomas J., Glowatzki, Elisabeth, Fuchs, Paul Albert, McIntosh, J. Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8354143/
https://www.ncbi.nlm.nih.gov/pubmed/34385908
http://dx.doi.org/10.3389/fncel.2021.697560
_version_ 1783736540404383744
author Fisher, Fernando
Zhang, Yuanyuan
Vincent, Philippe F. Y.
Gajewiak, Joanna
Gordon, Thomas J.
Glowatzki, Elisabeth
Fuchs, Paul Albert
McIntosh, J. Michael
author_facet Fisher, Fernando
Zhang, Yuanyuan
Vincent, Philippe F. Y.
Gajewiak, Joanna
Gordon, Thomas J.
Glowatzki, Elisabeth
Fuchs, Paul Albert
McIntosh, J. Michael
author_sort Fisher, Fernando
collection PubMed
description Efferent cholinergic neurons inhibit sensory hair cells of the vertebrate inner ear through the combined action of calcium-permeable α9α10-containing nicotinic acetylcholine receptors (nAChRs) and associated calcium-dependent potassium channels. The venom of cone snails is a rich repository of bioactive peptides, many with channel blocking activities. The conopeptide analog, RgIA-5474, is a specific and potent antagonist of α9α10-containing nAChRs. We added an alkyl functional group to the N-terminus of the RgIA-5474, to enable click chemistry addition of the fluorescent cyanine dye, Cy3. The resulting peptide, Cy3-RgIA-5727, potently blocked mouse α9α10 nAChRs expressed in Xenopus oocytes (IC(50) 23 pM), with 290-fold less activity on α7 nAChRs and 40,000-fold less activity on all other tested nAChR subtypes. The tight binding of Cy3-RgIA-5727 provided robust visualization of hair cell nAChRs juxtaposed to cholinergic efferent terminals in excised, unfixed cochlear tissue from mice. Presumptive postsynaptic sites on outer hair cells (OHCs) were labeled, but absent from inner hair cells (IHCs) and from OHCs in cochlear tissue from α9-null mice and in cochlear tissue pre-incubated with non-Cy3-conjugated RgIA-5474. In cochlear tissue from younger (postnatal day 10) mice, Cy3-RgIA-5727 also labeled IHCs, corresponding to transient efferent innervation at that age. Cy3 puncta in Kölliker’s organ remained in the α9-null tissue. Pre-exposure with non-Cy3-conjugated RgIA-5474 or bovine serum albumin reduced this non-specific labeling to variable extents in different preparations. Cy3-RgIA-5727 and RgIA-5474 blocked the native hair cell nAChRs, within the constraints of application to the excised cochlear tissue. Cy3-RgIA-5727 or RgIA-5474 block of efferent synaptic currents in young IHCs was not relieved after 50 min washing, so effectively irreversible.
format Online
Article
Text
id pubmed-8354143
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-83541432021-08-11 Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells Fisher, Fernando Zhang, Yuanyuan Vincent, Philippe F. Y. Gajewiak, Joanna Gordon, Thomas J. Glowatzki, Elisabeth Fuchs, Paul Albert McIntosh, J. Michael Front Cell Neurosci Neuroscience Efferent cholinergic neurons inhibit sensory hair cells of the vertebrate inner ear through the combined action of calcium-permeable α9α10-containing nicotinic acetylcholine receptors (nAChRs) and associated calcium-dependent potassium channels. The venom of cone snails is a rich repository of bioactive peptides, many with channel blocking activities. The conopeptide analog, RgIA-5474, is a specific and potent antagonist of α9α10-containing nAChRs. We added an alkyl functional group to the N-terminus of the RgIA-5474, to enable click chemistry addition of the fluorescent cyanine dye, Cy3. The resulting peptide, Cy3-RgIA-5727, potently blocked mouse α9α10 nAChRs expressed in Xenopus oocytes (IC(50) 23 pM), with 290-fold less activity on α7 nAChRs and 40,000-fold less activity on all other tested nAChR subtypes. The tight binding of Cy3-RgIA-5727 provided robust visualization of hair cell nAChRs juxtaposed to cholinergic efferent terminals in excised, unfixed cochlear tissue from mice. Presumptive postsynaptic sites on outer hair cells (OHCs) were labeled, but absent from inner hair cells (IHCs) and from OHCs in cochlear tissue from α9-null mice and in cochlear tissue pre-incubated with non-Cy3-conjugated RgIA-5474. In cochlear tissue from younger (postnatal day 10) mice, Cy3-RgIA-5727 also labeled IHCs, corresponding to transient efferent innervation at that age. Cy3 puncta in Kölliker’s organ remained in the α9-null tissue. Pre-exposure with non-Cy3-conjugated RgIA-5474 or bovine serum albumin reduced this non-specific labeling to variable extents in different preparations. Cy3-RgIA-5727 and RgIA-5474 blocked the native hair cell nAChRs, within the constraints of application to the excised cochlear tissue. Cy3-RgIA-5727 or RgIA-5474 block of efferent synaptic currents in young IHCs was not relieved after 50 min washing, so effectively irreversible. Frontiers Media S.A. 2021-07-23 /pmc/articles/PMC8354143/ /pubmed/34385908 http://dx.doi.org/10.3389/fncel.2021.697560 Text en Copyright © 2021 Fisher, Zhang, Vincent, Gajewiak, Gordon, Glowatzki, Fuchs and McIntosh. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Neuroscience
Fisher, Fernando
Zhang, Yuanyuan
Vincent, Philippe F. Y.
Gajewiak, Joanna
Gordon, Thomas J.
Glowatzki, Elisabeth
Fuchs, Paul Albert
McIntosh, J. Michael
Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells
title Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells
title_full Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells
title_fullStr Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells
title_full_unstemmed Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells
title_short Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells
title_sort cy3-rgia-5727 labels and inhibits α9-containing nachrs of cochlear hair cells
topic Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8354143/
https://www.ncbi.nlm.nih.gov/pubmed/34385908
http://dx.doi.org/10.3389/fncel.2021.697560
work_keys_str_mv AT fisherfernando cy3rgia5727labelsandinhibitsa9containingnachrsofcochlearhaircells
AT zhangyuanyuan cy3rgia5727labelsandinhibitsa9containingnachrsofcochlearhaircells
AT vincentphilippefy cy3rgia5727labelsandinhibitsa9containingnachrsofcochlearhaircells
AT gajewiakjoanna cy3rgia5727labelsandinhibitsa9containingnachrsofcochlearhaircells
AT gordonthomasj cy3rgia5727labelsandinhibitsa9containingnachrsofcochlearhaircells
AT glowatzkielisabeth cy3rgia5727labelsandinhibitsa9containingnachrsofcochlearhaircells
AT fuchspaulalbert cy3rgia5727labelsandinhibitsa9containingnachrsofcochlearhaircells
AT mcintoshjmichael cy3rgia5727labelsandinhibitsa9containingnachrsofcochlearhaircells