Abstract

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 (InsP₃)-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 InsP₃receptors 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⁺]_irise 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.