Greeson Jennifer N, Raphael Robert M
Rice University, Department of Bioengineering, Houston, Texas 77251-1892, USA.
Biophys J. 2009 Jan;96(2):510-20. doi: 10.1016/j.bpj.2008.09.016.
Outer hair cell (OHC) electromotility enables frequency selectivity and sensitivity in mammalian audition. Electromotility is generated by the transmembrane protein prestin and is sensitive to amphipathic compounds including salicylate, chlorpromazine (CPZ), and trinitrophenol (TNP). Although these compounds induce observable membrane curvature changes in erythrocytes, their effects on OHC membrane curvature are unknown. In this work, fluorescence polarization microscopy was applied to investigate the effects of salicylate, CPZ, and TNP on di-8-ANEPPS orientation in the OHC plasma membrane. Our results demonstrate the ability of fluorescence polarization microscopy to measure amphipath-induced changes in di-8-ANEPPS orientation, consistent with nanoscale changes in membrane curvature between regularly spaced proteins connecting the OHC plasma membrane and cytoskeleton. Simultaneous application of oppositely charged amphipaths generally results in no net membrane bending, consistent with predictions of the bilayer couple hypothesis; however, the application of salicylate (10 mM), which inhibits electromotility, is not reversed by the addition of CPZ. This result supports other findings that suggest salicylate primarily influences electromotiliy and OHC nonlinear capacitance via a direct interaction with prestin. In contrast, we find that CPZ and TNP influence the voltage sensitivity of prestin via membrane bending, demonstrating the mechanosensitivity of this unique membrane motor protein.
外毛细胞（OHC）的电运动性使哺乳动物听觉具有频率选择性和敏感性。电运动性由跨膜蛋白prestin产生，并且对包括水杨酸盐、氯丙嗪（CPZ）和三硝基苯酚（TNP）在内的两亲性化合物敏感。尽管这些化合物在红细胞中会引起可观察到的膜曲率变化，但它们对OHC膜曲率的影响尚不清楚。在这项工作中，应用荧光偏振显微镜来研究水杨酸盐、CPZ和TNP对OHC质膜中di-8-ANEPPS取向的影响。我们的结果证明了荧光偏振显微镜测量两亲性物质诱导的di-8-ANEPPS取向变化的能力，这与连接OHC质膜和细胞骨架的规则间隔蛋白质之间膜曲率的纳米级变化一致。同时应用带相反电荷的两亲性物质通常不会导致净膜弯曲，这与双层偶联假说的预测一致；然而，抑制电运动性的水杨酸盐（10 mM）的应用不会因添加CPZ而逆转。这一结果支持了其他研究结果，即水杨酸盐主要通过与prestin的直接相互作用来影响电运动性和OHC非线性电容。相比之下，我们发现CPZ和TNP通过膜弯曲影响prestin的电压敏感性，证明了这种独特的膜运动蛋白的机械敏感性。
