ATP-Induced Ca²⁺ Release in Cochlear Outer Hair Cells: Localization of an Inositol Triphosphate-Gated Ca²⁺ Store to the Base of the Sensory Hair Bundle

Abstract

We used a high-performance fluorescence imaging system to visualize rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) evoked by focal applications of extracellular ATP to the hair bundle of outer hair cells (OHCs): the sensory-motor receptors of the cochlea. Simultaneous recordings of the whole-cell current and Calcium Green-1 fluorescence showed a two-component increase in [Ca(2+)](i). After an initial entry of Ca(2+) through the apical membrane, a second and larger, inositol triphosphate (InsP(3))-gated, [Ca(2+)](i) surge occurred at the base of the hair bundle. Electron microscopy of this intracellular Ca(2+) release site showed that it coincides with the localization of a unique system of endoplasmic reticulum (ER) membranes and mitochondria known as Hensen's body. Using confocal immunofluorescence microscopy, we showed that InsP(3) receptors share this location. Consistent with a Ca(2+)-mobilizing second messenger system linked to ATP-P2 receptors, we also determined that an isoform of G-proteins is present in the stereocilia. Voltage-driven cell shape changes and nonlinear capacitance were monitored before and after ATP application, showing that the ATP-evoked [Ca(2+)](i) rise did not interfere with the OHC electromotility mechanism. This second messenger signaling mechanism bypasses the Ca(2+)-clearance power of the stereocilia and transiently elevates [Ca(2+)](i) at the base of the hair bundle, where it can potentially modulate the action of unconventional myosin isozymes involved in maintaining the hair bundle integrity and potentially influence mechanotransduction.

Differential expression of P2Y receptors in the rat cochlea during development

“…As noted, P2X 2 receptors are confined to the apical surface, particularly the stereocilia of the hair cells [41,52], while it seems likely that P2X 7 receptor protein is localized on the basolateral surface of the outer hair cells [53] and may influence prestin-mediated electromotility [37]. Ion imaging has indicated that extracellular ATP invokes apically mediated Ca 2+ entry, via the P2X 2 receptors [54,55], however, this is complemented by apically localized P2Y receptor signaling that causes a release of Ca 2+ stored within Hensen's body, in the cuticular plate region of the outer hair cells, via the PLC-IP 3 pathway [55]. Our data indicate that this would likely be mediated by both P2Y 2 and P2Y 6 receptors.…”

“…Synthesis of membrane-bound DAG following activation of these P2Y receptors, may also trigger Ca 2+ entry via colocalized canonical transient receptor potential channels (TRPC3) [56,57]. This localized Ca 2+ influx may influence the mechanoelectrical transducer channel conductance [55], or more generally, the multi-modal cation influx may affect neurotransmission, and for outer hair cells, voltagedependent electromotility.…”

ATP activates P2x receptors and requires extracellular Ca++ participation to modify outer hair cell nonlinear capacitance

“…P2x2 is a predominant isoform expressed at the stereocilia and the cuticular plate [10,16,22,23]. It has been further hypothesized that the application of ATP may also result in the diffusion of a G protein to a specialized inositol 1,4,5-trisphosphate (IP 3 ) receptor-gated Ca ++ store (Hensen's body) present beneath the cuticular plate to raise intracellular Ca ++ and affect the mechanoelectrical conductance, particularly the adaptation rate [35]. In this experiment, the chelation of intracellular Ca ++ by a fast Ca ++ chelator BAPTA did not alter the effect of ATP on OHC electromotility (Figs.…”

Role of nitric oxide on purinergic signalling in the cochlea

“…ATP has been described as a neurotransmitter or a neuromodulator in auditory neurotransmission. ATP has been reported to induce an increase of intracellular Ca 2+ concentration ([Ca 2+ ] i ) in outer hair cells (OHCs) [4,5], inner hair cells (IHCs) [6][7][8], spiral ganglion neurons (SGNs) [9,10], and supporting cells [11][12][13]. These previous studies suggested that ATP may affect auditory neurotransmission by modulating [Ca 2+ ] i in the cochlear cells.…”

“…These previous studies suggested that ATP may affect auditory neurotransmission by modulating [Ca 2+ ] i in the cochlear cells. It has been shown that OHCs and IHCs possess both ligand-gated ionotropic P2X receptors and G proteincoupled metabotropic P2Y receptors [5,6,14]. The extensive distribution of P2X 2 receptor subunit expression in the guinea pig cochlea provides evidence for divergent roles for extracellular ATP acting via ATP-gated ion channels [14].…”