Alansary Dalia, Kilch Tatiana, Holzmann Christian, Peinelt Christine, Hoth Markus, Lis Annette
Department of Biophysics, Saarland University, Homburg, Germany.
Cold Spring Harb Protoc. 2014 Jun 2;2014(6):602-7. doi: 10.1101/pdb.top066795.
Depletion of internal Ca(2+) stores activates store-operated Ca(2+) channels. The most prominent members of this class of channels are Ca(2+) release-activated Ca(2+) (CRAC) channels, which are present in a variety of cell types including immune cells. CRAC channels are composed of ORAI proteins, which are activated by endoplasmic reticulum-bound STIM proteins on Ca(2+) store depletion. The underlying Ca(2+) current is called ICRAC, which is required for many cellular functions including T-cell activation, mast cell activation, Ca(2+)-dependent gene expression, and refilling of internal Ca(2+) stores. To analyze ICRAC or the Ca(2+) current through heterologously expressed ORAI channels, whole-cell patch clamp is the technique of choice. It allows the direct analysis of ion currents through CRAC/ORAI channels. The patch-clamp technique has been used to determine selectivity, permeability, rectification, inactivation, and several other biophysical and pharmacological properties of the channels, and is the most direct and reliable technique to analyze ICRAC.
细胞内钙离子储存的耗竭会激活储存操纵性钙离子通道。这类通道中最突出的成员是钙离子释放激活钙离子（CRAC）通道，其存在于包括免疫细胞在内的多种细胞类型中。CRAC通道由ORAI蛋白组成，在钙离子储存耗竭时，ORAI蛋白会被内质网结合的STIM蛋白激活。其潜在的钙离子电流称为ICRAC，许多细胞功能都需要ICRAC，包括T细胞激活、肥大细胞激活、钙离子依赖的基因表达以及细胞内钙离子储存的再填充。为了分析ICRAC或通过异源表达的ORAI通道的钙离子电流，全细胞膜片钳是首选技术。它可以直接分析通过CRAC/ORAI通道的离子电流。膜片钳技术已被用于确定通道的选择性、通透性、整流、失活以及其他一些生物物理和药理学特性，并且是分析ICRAC最直接、最可靠的技术。
