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

We used a high-performance fluorescence imaging system to visualize rapid c hanges in intracellular free Ca2+ concentration ([Ca2+](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-com ponent increase in [Ca2+](i). After an initial entry of Ca2+ through the ap ical membrane, a second and larger, inositol triphosphate (InsP(3))-gated, [Ca2+](i) surge occurred at the base of the hair bundle. Electron microscop y of this intracellular Ca2+ release site showed that it coincides with the localization of a unique system of endoplasmic reticulum (ER) membranes an d mitochondria known as Hensen's body. Using confocal immunofluorescence mi croscopy, we showed that InsP(3) receptors share this location. Consistent with a Ca2+-mobilizing second messenger system linked to ATP-P2 receptors, we also determined that an isoform of G-proteins is present in the stereoci lia. Voltage-driven cell shape changes and nonlinear capacitance were monit ored before and after ATP application, showing that the ATP-evoked [Ca2+](i ) rise did not interfere with the OHC electromotility mechanism. This secon d messenger signaling mechanism bypasses the Ca2+-clearance power of the st ereocilia and transiently elevates [Ca2+](i) at the base of the hair bundle , where it can potentially modulate the action of unconventional myosin iso zymes involved in maintaining the hair bundle integrity and potentially inf luence mechanotransduction.