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

Fernando Fisher,Elisabeth Glowatzki,Paul Albert Fuchs,Thomas J Gordon,Joanna Gajewiak,Philippe F Y Vincent,J Michael Mcintosh,Yuanyuan Zhang

doi:10.3389/fncel.2021.697560

Abstract

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.

Highlights

Mammalian nicotinic acetylcholine receptors are pentameric ligand gated ion channels that are used for neurotransmission throughout the nervous system
Subtype selectivity was assessed by measuring the activity at other nicotinic acetylcholine receptors (nAChRs) subtypes
Recordings exemplify results obtained from five inner hair cells (IHCs) from five mice. (B) Reduction of efferent postsynaptic current over time in the presence of 10 nM RgIA-5474 or 10 nM Cy3-RgIA-5727

Summary

Introduction

Mammalian nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels that are used for neurotransmission throughout the nervous system. Cone snails are predatory marine organisms that employ complex mixtures of venom peptides. Sensory hair cells are cilia-like structures that convert mechanical energy into electrical signals. Such hair cells are present in marine organisms and used to detect motion, pressure, and vibration of surrounding water (Fritzsch and Straka, 2014). For example, have hair cells arranged in a mechanosensory lateral line organ that is used, in part, to avoid capture. It was demonstrated that these lateral line hair cells utilize α9 nAChRs at the efferent synapse to mediate ACh neurotransmission (Carpaneto Freixas et al, 2021)

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Results

Conclusion