基于荧光的比率测量法检测过表达STIM和Orai的HEK-293细胞中CRAC通道活性

Fluorescence-Based Ratiometric Measurement of CRAC Channel Activity in STIM-Orai-Overexpressing HEK-293 Cells.

作者信息

Zhang Shuce, He Lian, Zhou Yubin, Wang Youjun

机构信息

Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, China.

Department of Medical Physiology, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, USA.

出版信息

Methods Mol Biol. 2018;1843:17-39. doi: 10.1007/978-1-4939-8704-7_2.

DOI:10.1007/978-1-4939-8704-7_2

PMID:30203274

Abstract

Calcium influx through store-operated Ca entry (SOCE), mediated by STIM-operated Orai channels, is crucial for many cellular functions. To dissect the molecular mechanisms underlying the process of STIM-Orai activation and identify regulators that modify this process, ratiometric imaging of SOCE responses in HEK cells overexpressing STIM and Orai is a routinely used method. Here we describe one commonly used procedure of monitoring SOCE activity with a ratiometric membrane-permeable dye fura-2-AM. Other ratiometric indicators suitable for SOCE measurements are also discussed.

摘要

由STIM激活的Orai通道介导的通过储存操纵性钙内流（SOCE）的钙内流对许多细胞功能至关重要。为了剖析STIM-Orai激活过程的分子机制并鉴定修饰该过程的调节因子，在过表达STIM和Orai的HEK细胞中对SOCE反应进行比率成像分析是一种常用方法。在此，我们描述一种使用比率性膜通透性染料fura-2-AM监测SOCE活性的常用程序。还讨论了其他适用于SOCE测量的比率性指示剂。