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...
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Frontiers Media S.A.
2021-07-01
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| Series: | Frontiers in Cellular Neuroscience |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fncel.2021.697560/full |
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| author | Fernando Fisher Yuanyuan Zhang Philippe F. Y. Vincent Joanna Gajewiak Thomas J. Gordon Elisabeth Glowatzki Paul Albert Fuchs J. Michael McIntosh J. Michael McIntosh J. Michael McIntosh |
| author_facet | Fernando Fisher Yuanyuan Zhang Philippe F. Y. Vincent Joanna Gajewiak Thomas J. Gordon Elisabeth Glowatzki Paul Albert Fuchs J. Michael McIntosh J. Michael McIntosh J. Michael McIntosh |
| author_sort | Fernando Fisher |
| collection | DOAJ |
| 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 (IC50 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. |
| first_indexed | 2024-12-14T21:52:20Z |
| format | Article |
| id | doaj.art-387b50ea22dd45509aeb0bc2aefdcc6c |
| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-12-14T21:52:20Z |
| publishDate | 2021-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular Neuroscience |
| spelling | doaj.art-387b50ea22dd45509aeb0bc2aefdcc6c2022-12-21T22:46:12ZengFrontiers Media S.A.Frontiers in Cellular Neuroscience1662-51022021-07-011510.3389/fncel.2021.697560697560Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair CellsFernando Fisher0Yuanyuan Zhang1Philippe F. Y. Vincent2Joanna Gajewiak3Thomas J. Gordon4Elisabeth Glowatzki5Paul Albert Fuchs6J. Michael McIntosh7J. Michael McIntosh8J. Michael McIntosh9Department of Biology, University of Utah, Salt Lake City, UT, United StatesThe Center for Hearing and Balance, Otolaryngology-Head and Neck Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Center for Hearing and Balance, Otolaryngology-Head and Neck Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesDepartment of Biology, University of Utah, Salt Lake City, UT, United StatesDepartment of Biology, University of Utah, Salt Lake City, UT, United StatesThe Center for Hearing and Balance, Otolaryngology-Head and Neck Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Center for Hearing and Balance, Otolaryngology-Head and Neck Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesDepartment of Biology, University of Utah, Salt Lake City, UT, United StatesGeorge E. Wahlen Department of Veterans Affairs Medical Center, Salt Lake City, UT, United StatesDepartment of Psychiatry, University of Utah School Medicine, Salt Lake City, UT, United StatesEfferent 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 (IC50 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.https://www.frontiersin.org/articles/10.3389/fncel.2021.697560/fullconotoxinnAChRcochleamouseXenopus |
| spellingShingle | Fernando Fisher Yuanyuan Zhang Philippe F. Y. Vincent Joanna Gajewiak Thomas J. Gordon Elisabeth Glowatzki Paul Albert Fuchs J. Michael McIntosh J. Michael McIntosh J. Michael McIntosh Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells Frontiers in Cellular Neuroscience conotoxin nAChR cochlea mouse Xenopus |
| 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 | conotoxin nAChR cochlea mouse Xenopus |
| url | https://www.frontiersin.org/articles/10.3389/fncel.2021.697560/full |
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