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Front Cell Neurosci
2021 Jan 01;15:697560. doi: 10.3389/fncel.2021.697560.
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Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells.
Fisher F
,
Zhang Y
,
Vincent PFY
,
Gajewiak J
,
Gordon TJ
,
Glowatzki E
,
Fuchs PA
,
McIntosh JM
.
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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.
FIGURE 1. Synthesis and HPLC purification of Cy3-RgIA-5727. (A) The N-alkyne-containing RgIA analog was reacted with Cy3-azide, in the presence of Cu (I) as a catalyst, to form Cy3-RgIA-5727. (B) Chromatography profile of alkyne-bearing precursor RgIA-5727. (C) Chromatography profile of the crude click reaction mixture. The major peak corresponds to the desired product, Cy3-RgIA-5727.
FIGURE 2. Block and recovery from block of α9α10 nAChRs by Cy3-RgIA-5727. (A) Cy3-RgIA-5727 (10 nM) was applied to Xenopus oocytes expressing mouse α9α10 nAChRs as described in Section âMaterials and Methods.â Recovery from block after removal of peptide is shown. (B) Concentration response analysis indicated an IC50 of 23 pM (see Table 1); n = 3â4 oocytes. (C) Time course of block and (D) un-block by 10 nM Cy3-RgIA-5727; n = 3 oocytes.
FIGURE 3. Block and recovery from block of α7 nAChRs by Cy3 RgIA-5727. (A) Cy3-RgIA-5727 (10 nM) was perfusion applied to Xenopus laevis oocytes expressing mouse α7 nAChRs. Note the rapid recovery from block after removal of peptide. (B) Concentration response analysis indicated an IC50 of 6.8 nM (see Table 1).
Ahmad Fuaad,
Peptide conjugation via CuAAC 'click' chemistry.
2013, Pubmed
Ahmad Fuaad,
Peptide conjugation via CuAAC 'click' chemistry.
2013,
Pubmed
Carpaneto Freixas,
Unraveling the Molecular Players at the Cholinergic Efferent Synapse of the Zebrafish Lateral Line.
2021,
Pubmed
Christensen,
RgIA4 Potently Blocks Mouse α9α10 nAChRs and Provides Long Lasting Protection against Oxaliplatin-Induced Cold Allodynia.
2017,
Pubmed
,
Xenbase
Dutertre,
Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails.
2014,
Pubmed
Elgoyhen,
Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells.
1994,
Pubmed
,
Xenbase
Elgoyhen,
The nicotinic receptor of cochlear hair cells: a possible pharmacotherapeutic target?
2009,
Pubmed
Ellison,
Alpha-RgIA: a novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR.
2006,
Pubmed
,
Xenbase
Fritzsch,
Evolution of vertebrate mechanosensory hair cells and inner ears: toward identifying stimuli that select mutation driven altered morphologies.
2014,
Pubmed
Gajewiak,
Selective Penicillamine Substitution Enables Development of a Potent Analgesic Peptide that Acts through a Non-Opioid-Based Mechanism.
2021,
Pubmed
Glowatzki,
Cholinergic synaptic inhibition of inner hair cells in the neonatal mammalian cochlea.
2000,
Pubmed
Goutman,
Facilitating efferent inhibition of inner hair cells in the cochlea of the neonatal rat.
2005,
Pubmed
Hecker,
Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1β Release.
2015,
Pubmed
Hiel,
Expression of nicotinic acetylcholine receptor mRNA in the adult rat peripheral vestibular system.
1996,
Pubmed
Hone,
Computational and Functional Mapping of Human and Rat α6β4 Nicotinic Acetylcholine Receptors Reveals Species-Specific Ligand-Binding Motifs.
2021,
Pubmed
,
Xenbase
Jin,
Conotoxins: Chemistry and Biology.
2019,
Pubmed
Katz,
Developmental regulation of nicotinic synapses on cochlear inner hair cells.
2004,
Pubmed
Kolb,
Click Chemistry: Diverse Chemical Function from a Few Good Reactions.
2001,
Pubmed
Liberman,
Afferent and efferent innervation of the cat cochlea: quantitative analysis with light and electron microscopy.
1990,
Pubmed
Luo,
Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist.
2015,
Pubmed
,
Xenbase
McIntosh,
A novel alpha-conotoxin, PeIA, cloned from Conus pergrandis, discriminates between rat alpha9alpha10 and alpha7 nicotinic cholinergic receptors.
2005,
Pubmed
,
Xenbase
Morley,
Identification of the subunits of the nicotinic cholinergic receptors in the rat cochlea using RT-PCR and in situ hybridization.
1998,
Pubmed
Muttenthaler,
On-resin strategy to label α-conotoxins: Cy5-RgIA, a potent α9α10 nicotinic acetylcholine receptor imaging probe.
2020,
Pubmed
,
Xenbase
Nadol,
Supranuclear efferent synapses on outer hair cells and Deiters' cells in the human organ of Corti.
1994,
Pubmed
Peng,
Characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) and alpha (alpha) 10 (CHRNA10) in lymphocytes.
2004,
Pubmed
Richter,
Phosphocholine - an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors.
2016,
Pubmed
,
Xenbase
Richter,
Amyloid Beta Peptide (Aβ1-42) Reverses the Cholinergic Control of Monocytic IL-1β Release.
2020,
Pubmed
Richter,
C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation.
2018,
Pubmed
Romero,
Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain.
2017,
Pubmed
Roux,
Onset of cholinergic efferent synaptic function in sensory hair cells of the rat cochlea.
2011,
Pubmed
Siebers,
Alpha-1 Antitrypsin Inhibits ATP-Mediated Release of Interleukin-1β via CD36 and Nicotinic Acetylcholine Receptors.
2018,
Pubmed
Simmons,
Development of the inner ear efferent system across vertebrate species.
2002,
Pubmed
Simmons,
Differential expression of the alpha 9 nicotinic acetylcholine receptor subunit in neonatal and adult cochlear hair cells.
1998,
Pubmed
Vattino,
Functional Postnatal Maturation of the Medial Olivocochlear Efferent-Outer Hair Cell Synapse.
2020,
Pubmed
Vetter,
Role of alpha9 nicotinic ACh receptor subunits in the development and function of cochlear efferent innervation.
1999,
Pubmed
Vyas,
Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea.
2020,
Pubmed
Zakrzewicz,
SLPI Inhibits ATP-Mediated Maturation of IL-1β in Human Monocytic Leukocytes: A Novel Function of an Old Player.
2019,
Pubmed
,
Xenbase
Zhang,
Nicotine evoked efferent transmitter release onto immature cochlear inner hair cells.
2020,
Pubmed
