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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 “Materials and Methods.” Recovery from block after removal of peptide is shown. (B) Concentration response analysis indicated an IC50 of 23 pM (see Table 1); n = 3–4 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).
FIGURE 4. Cy3-RgIA-5727 label in Wild Type P14 mouse cochlea. At this age efferent synapses are found on OHCs with little to none of the transient efferent innervation to IHCs. Cy3-RgIA-5727 (10 nM) produced red puncta associated with OHCs (labeled green by accumulated FM dye). (A) Maximum intensity projection (MIP) from the apical surface of the cochlea. (B) Optical yz-plane view of cochlea (MIP), three rows of OHCs on left, IHCs on right, green FM4-64 accumulation fills hair cell somata and stereocilia. Note that the MIP shows multiple cells in each position. (C) Cy3-RgIA-5727 in yz-plane view. Large number of puncta on OHCs captured by MIP that includes approximately 10 hair cells in each position. FM4-64 fluorescence bleeds through to label stereocilia faintly. Additional puncta observed modiolar to the IHCs in region of greater epithelial ridge, but not along the synaptic pole of the IHCs. (D) Merged view of FM4-64 and Cy3-RgIA-5727 (MIP). Note that red Cy3 puncta do not have corresponding green puncta in panel (B). Inner hair cell (IHC) and greater epithelial ridge (GER) outlined with dotted lines. Images exemplify results obtained from four cochleas from four mice. See Supplementary Video 1.
FIGURE 5. Cy3-RgIA-5727 label in Wild Type P10 cochlea. This age is near the peak of MOC innervation onto IHCs, and innervation of OHCs is well under way. (A) Maximum intensity projection (MIP) from the apical surface of the cochlea. (B) Optical yz-plane view of cochlea (MIP), three rows of OHCs on left, single row of IHCs on right, green FM4-64 accumulation fills multiple hair cells seen in depth. (C) Cy3-RgIA-5727 in yz-plane view (MIP); red puncta observed on OHCs and IHCs. (D) Merged view of FM4-64 and Cy3-RgIA-5727 (MIP). Red Cy3 puncta readily distinguished against green FM4-64 background. Inner hair cell (IHC) and greater epithelial ridge (GER) outlined with dashed lines. Images exemplify results from 12 cochleas from 12 mice. See Supplementary Video 2.
FIGURE 6. Cochlear location of Cy3-RgIA-5727 puncta. (A) Cy3 puncta associated with hair cells were counted in cochleas from C57 Bl6 mice before (P10) and after the onset of hearing (P14). (B) Cochleas from P23 mice expressing the fluorescent protein m-citrine under the ChAT promoter (Chat-Cre) were excised and exposed to Cy3-RgIA-5727 in the presence of 0.1 mg/ml BSA. Cy3-puncta (red) were found under OHCs, but not under IHCs. Note also the absence of Cy3 puncta medial to the IHCs. Cy3-puncta were closely aligned with m-citrine positive efferent terminals (green). See Supplementary Video 3 for 3-D view.
FIGURE 7. Cy3-RgIA-5727 labeling in P12 α9-null mouse cochlea. At this age efferent synapses occur on IHCs and OHCs, even in the absence of the α9 subunit. (A) Maximum intensity projection (MIP) from the apical surface of the cochlea. (B) Optical yz-plane view of cochlea (MIP), three rows of OHCs on left, IHCs on right, green FM4-64 accumulation. (C) Cy3-RgIA-5727 in yz-plane view (MIP). (D) Merged view of FM4-64 and Cy3-RgIA-5727 (MIP). No Cy3 puncta are found in the synaptic regions of IHCs or OHCs. Cy3-RgIA-5727 puncta occurred medial to the IHCs in the greater epithelial ridge [inset and white arrowheads in panel (A)]. Images exemplify results obtained from two cochleas from two knockout mice. See Supplementary Video 4.
FIGURE 8. Cy3-RgIA-5727 labeling in P10 WT mouse cochlea pre-exposed to non-conjugated RgIA-5474. At this age one can expect efferent synapses on IHCs and OHCs. (A) Maximum intensity projection (MIP) from the apical surface of the cochlea. (B) Optical yz-plane view of cochlea (MIP), three rows of OHCs on left, single row of IHCs on right, green FM4-64 accumulation. (C) Cy3-RgIA-5727 in yz-plane (MIP). (D) Merged view of FM4-64 and Cy3-RgIA-5727 (MIP). No Cy3 puncta are found in the synaptic regions of IHCs (outlined with dashed lines) or OHCs but Cy3 puncta are seen in the greater epithelial ridge (GER, outlined with dashed lines). Images exemplify results obtained from two cochleas from two mice. See Supplementary Video 5.
FIGURE 9. Application of 10 nM Cy3-RgIA-5727 or RgIA-5474 gradually reduced the amplitude of efferent postsynaptic currents. (A) Synaptic current recorded at –90 mV in a P9 IHC during a 1 Hz train of 100 shocks. Each response is the average response at the indicated time point as Cy3-RgIA-5727 spread through the tissue. After washing out toxin for 45 min there was no recovery. Recordings exemplify results obtained from five IHCs from five mice. (B) Reduction of efferent postsynaptic current (normalized to initial pre-block value) over time in the presence of 10 nM RgIA-5474 (black) or 10 nM Cy3-RgIA-5727 (red). All data points collected at 9, 15, 20 min but displaced horizontally for clarity. Experimental data fitted with y=A1-A21+e(x-x0)/dx+A2, where A1 = initial value, A2 = final value, X0 = center value, dx = time constant. Control data from 10 other IHCs not subject to blockers (gray) are fit with a straight line. Blocked data significantly different from control. But there was no significant difference in time course or degree of block at 20 min between RgIA-5474 and Cy3-RgIA-5727. Inclusion of 0.1 mg/ml bovine serum albumin (BSA) prevented surface absorption of peptide and significantly accelerated block onset (green).
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