培养细胞中prestin的功能表达及微区定位
Functional expression and microdomain localization of prestin in cultured cells.
作者信息
Sturm Angela K, Rajagopalan Lavanya, Yoo Donald, Brownell William E, Pereira Fred A
机构信息
Bobby R. Alford Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, Houston, TX 77030, USA.
出版信息
Otolaryngol Head Neck Surg. 2007 Mar;136(3):434-9. doi: 10.1016/j.otohns.2006.10.030.
INTRODUCTION
Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane.
METHODS
We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature.
RESULTS & DISCUSSION: Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC.
CONCLUSION
Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.
引言
Prestin是耳蜗外毛细胞分子马达的重要组成部分，有助于哺乳动物听力的频率选择性和敏感性。研究Prestin的一个模型系统是将其转染到培养的HEK 293细胞中。我们的目标是表征Prestin的运输途径及其在质膜中的定位。
方法
我们使用Prestin与细胞内和质膜标记物的免疫共定位以及蔗糖密度梯度离心法来分析膜区室中的Prestin。采用电压钳位法测量非线性电容（NLC），即Prestin的电信号特征。
结果与讨论
转染后24小时，当可测量NLC时，Prestin靶向细胞膜。然后，Prestin聚集到与膜微区共定位并分级分离的膜灶中。耗尽膜胆固醇含量会改变Prestin的定位和NLC。
结论
HEK 293细胞中Prestin的活性源于其在质膜中的表达，改变膜脂质含量会影响Prestin的定位和活性。
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