MOLECULAR BASIS OF TRANSDUCTION IN AUDITORY SENSORY ORGANS

我们使用一个高性能的荧光成像系统来观察细胞外ATP对外毛细胞的毛束(耳蜗感觉-运动感受器)的集中作用所引起的细胞内游离钙离子浓度([Ca+]i)的快速变化。同时记录的全细胞电流和钙绿-1荧光显示，[Ca~(2+)]_i有两种成分的增加。在Ca~(2+)最初通过顶膜进入后，在毛束底部发生了第二次或更大的InsP3门控的[Ca~(2+)]_i波峰。这个细胞内钙离子释放部位的电子显微镜显示，它与一个独特的内质网膜和线粒体系统的定位相一致，即亨森氏小体。使用共聚焦免疫荧光显微镜，我们发现InsP3受体共享这个位置。与与ATP-P2受体相连的钙离子动员的第二信使系统相一致，我们还确定在立体纤毛中存在G蛋白的异构体。监测了ATP作用前后电压驱动的细胞形态变化和非线性电容变化，结果表明，ATP引起的[Ca~(2+)]i升高并不干扰OHC的电动机制。第二种信使信号机制绕过了静纤毛的钙清除能力，瞬时提高了毛束底部的[Ca~(2+)]i，在那里它可能调节参与维持毛束完整性的非传统肌球蛋白同工酶的活动，并可能影响机械转导。-毛发感觉细胞、机械转导、电动、耳蜗放大器、Corti器官、外毛细胞、嘌呤能受体。

We used a high-performance fluorescence imaging system to visualize rapid changes in intracellular free Ca2+ concentration ([Ca2+]i) evoked by focal applications of extracellullar 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, 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 Hensens body. Using confocal immunofluorescence microscopy, we showed that InsP3 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 mechano-transduction. - hair sensory cells, mechano transduction, electromotility, cochlear amplifier, organ of Corti, outer hair cells, purinergic receptors.