Xia Anping, Wooltorton Julian R A, Palmer Donna J, Ng Philip, Pereira Fred A, Eatock Ruth Anne, Oghalai John S
Bobby R. Alford Department of Otolaryngology, Head and Neck Surgery, Baylor College of Medicine, One Baylor Plaza, NA102, Houston, TX 77030, USA.
J Assoc Res Otolaryngol. 2008 Sep;9(3):307-20. doi: 10.1007/s10162-008-0121-3. Epub 2008 May 28.
Prestin is a membrane protein in the outer hair cell (OHC) that has been shown to be essential for electromotility. OHCs from prestin-null mice do not express prestin, do not have a nonlinear capacitance (the electrical signature of electromotility), and are smaller in size than wild-type OHCs. We sought to determine whether prestin-null OHCs can be transduced to incorporate functional prestin protein in a normal fashion. A recombinant helper-dependent adenovirus expressing prestin and green fluorescent protein (HDAd-prestin-GFP) was created and tested in human embryonic kidney cells (HEK cells). Transduced HEK cells demonstrated membrane expression of prestin and nonlinear capacitance. HDAd-prestin-GFP was then applied to cochlear sensory epithelium explants harvested from wild-type and prestin-null mice at postnatal days 2-3, the age at which native prestin is just beginning to become functional in wild-type mice. At postnatal days 4-5, we investigated transduced OHCs for (1) their prestin expression pattern as revealed by immunofluorescence; (2) their cell surface area as measured by linear capacitance; and (3) their prestin function as indicated by nonlinear capacitance. HDAd-prestin-GFP efficiently transduced OHCs of both genotypes and prestin protein localized to the plasma membrane. Whole-cell voltage clamp studies revealed a nonlinear capacitance in transduced wild-type and prestin-null OHCs, but not in non-transduced cells of either genotype. Prestin transduction did not increase the linear capacitance (cell surface area) for either genotype. In peak nonlinear capacitance, voltage at peak nonlinear capacitance, charge density of the nonlinear capacitance, and shape of the voltage-capacitance curves, the transduced cells of the two genotypes resembled each other and previously reported data from adult wild-type mouse OHCs. Thus, prestin introduced into prestin-deficient OHCs segregates normally to the cell membrane and generates a normal nonlinear capacitance, indicative of normal prestin function.
Prestin是外毛细胞（OHC）中的一种膜蛋白，已被证明对电运动至关重要。来自Prestin基因敲除小鼠的OHC不表达Prestin，没有非线性电容（电运动的电信号特征），并且尺寸比野生型OHC小。我们试图确定Prestin基因敲除的OHC是否可以被转导以正常方式整合功能性Prestin蛋白。构建了一种表达Prestin和绿色荧光蛋白的重组辅助依赖型腺病毒（HDAd-Prestin-GFP），并在人胚肾细胞（HEK细胞）中进行测试。转导的HEK细胞显示出Prestin的膜表达和非线性电容。然后将HDAd-Prestin-GFP应用于出生后2-3天从野生型和Prestin基因敲除小鼠收获的耳蜗感觉上皮外植体，此时野生型小鼠中的天然Prestin刚刚开始发挥功能。在出生后4-5天，我们研究转导的OHC的（1）免疫荧光显示的Prestin表达模式；（2）通过线性电容测量的细胞表面积；以及（3）由非线性电容指示的Prestin功能。HDAd-Prestin-GFP有效地转导了两种基因型的OHC，并且Prestin蛋白定位于质膜。全细胞电压钳研究显示转导的野生型和Prestin基因敲除的OHC中存在非线性电容，但两种基因型的未转导细胞中均未发现。Prestin转导并未增加两种基因型的线性电容（细胞表面积）。在峰值非线性电容、峰值非线性电容时的电压、非线性电容的电荷密度以及电压-电容曲线的形状方面，两种基因型的转导细胞彼此相似，并且与先前报道的成年野生型小鼠OHC的数据相似。因此，引入Prestin缺陷型OHC的Prestin正常地分离到细胞膜并产生正常的非线性电容，表明Prestin功能正常。
