ATP-induced Ca2+ release in cochlear outer hair cells: localization of an InsP3-gated Ca2+ store to the base of the sensory hair bundle.

We used a high-performance fluorescence imaging system to visualize rapid changes 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-component increase in [Ca2+](i). After an initial entry of Ca2+ through the apical membrane, a second and larger, inositol triphosphate (InsP(3))-gated, [Ca2+](i) surge occurred at the base of the hair bundle. Electron microscopy of this intracellular Ca2+ 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 Ca2+-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 [Ca2+](i) rise did not interfere with the OHC electromotility mechanism. This second messenger signaling mechanism bypasses the Ca2+-clearance power of the stereocilia and transiently elevates [Ca2+](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.