Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, USA.
Biophys J. 2010 Jul 7;99(1):85-94. doi: 10.1016/j.bpj.2010.03.066.
Prestin is a member of the SLC26 family of anion transporters and is responsible for electromotility in outer hair cells, the basis of cochlear amplification in mammals. It is an anion transporting transmembrane protein, possessing nine cysteine residues, which generates voltage-dependent charge movement. We determine the role these cysteine residues play in the voltage sensing capabilities of prestin. Mutations of any single cysteine residue had little or no effect on charge movement. However, using combinatorial substitution mutants, we identified a cysteine residue pair (C415 and either C192 or C196) whose mutation reduced or eliminated charge movement. Furthermore, we show biochemically that surface expression of mutants with markedly reduced functionality can be near normal; however, we identify two monomers of the protein on the surface of the cell, the larger of which correlates with surface charge movement. Because we showed previously by Förster resonance energy transfer that monomer interactions are required for charge movement, we tested whether disulfide interactions were required for dimerization. Using Western blots to detect oligomerization of the protein in which variable numbers of cysteines up to and including all nine cysteine residues were mutated, we show that disulfide bond formation is not essential for dimer formation. Taken together, we believe these data indicate that intramembranous cysteines are constrained, possibly via disulfide bond formation, to ensure structural features of prestin required for normal voltage sensing and mechanical activity.
Prestin 是 SLC26 家族阴离子转运体的成员，负责哺乳动物耳蜗放大的基础——外毛细胞的电运动。它是一种阴离子跨膜转运蛋白，含有 9 个半胱氨酸残基，可产生电压依赖性电荷移动。我们确定这些半胱氨酸残基在 prestin 的电压感应能力中的作用。单个半胱氨酸残基的突变几乎没有或没有影响电荷移动。然而，通过组合取代突变体，我们鉴定了一对半胱氨酸残基（C415 和 C192 或 C196），其突变降低或消除了电荷移动。此外，我们还从生物化学角度表明，功能明显降低的突变体的表面表达可以接近正常；然而，我们在细胞表面鉴定出两种蛋白单体，其中较大的单体与表面电荷移动相关。因为我们之前通过Förster 共振能量转移表明单体相互作用是电荷移动所必需的，所以我们测试了二硫键相互作用是否对二聚体形成是必需的。通过 Western blot 检测其中可变数量的半胱氨酸（多达和包括所有 9 个半胱氨酸残基）发生突变的蛋白的寡聚化，我们表明二硫键形成不是二聚体形成所必需的。综上所述，我们认为这些数据表明跨膜半胱氨酸受到约束，可能通过二硫键形成，以确保 prestin 的正常电压感应和机械活性所需的结构特征。
