The CRAC Channel, 1st ed. 2018 Methods and Protocols Methods in Molecular Biology Series, Vol. 1843

This volume discusses the cellular and molecular techniques used to study and characterize the different components of the CRAC channel signaling pathway. The chapters in this book cover topics such as fluorescence-based measurements of the CRAC channel activity in cell populations; patch-clamp recording of the CRAC channel in STIM-Orai overexpressing cells and native systems; western-blotting and co-immunoprecipitation of endogenous STIM/ORAI and protein partners; shRNA-mediated gene silencing; and the SC-SMD system. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Thorough and cutting-edge, The CRAC Channel: Methods and Protocols is a valuable resource for any researcher interested in learning more about the CRAC channel, as its activation mechanism and roles in cellular functions have yet to be completely explored.

Table of Contents…

Contributing Authors…

1. Patch-Clamp Recording of the CRAC Channel Current in STIM-Orai Overexpressing Cells

Yiming Zhang, Zheng Sisi, Yubin Zhou, Donald L. Gill, and Youjun Wang

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

Shuce Zhang, Lian He, Yubin Zhou, and Youjun Wang

3. Recording SOCE Activity in Neurons by Patch-Clamp Electrophysiology and Microfluorometric Calcium Imaging

Hsiang-en Wu, Geza Gemes, and Quinn H. Hogan

4. Mn2+ Quenching Assay for Store-Operated Calcium Entry

Zui Pan, Sangyong Choi, and Yanhong Luo

5. Fluorescence-Based Measurements of Store-Operated Ca2+ Entry in Cancer Cells using Fluo-4 and Confocal Live Cell Imaging

Fujian Lu, Jianwei Sun, Tao Sun, Heping Cheng, and Shengyu Yang

6. Fluorescence-Based Measurements of the CRAC Channel Activity in Cell Populations

Pedro C. Redondo, Alejandro Berna-Erro, Natalia Dionisio, and Juan A. Rosado

7. Indirect Measurement of CRAC Channel Activity using NFAT Nuclear Translocation by Flow Cytometry in Jurkat Cells

María D. Carretta, María A, Hidalgo, and Rafael A. Burgos

8. CRAC Channel  Components Quantitative Expression (In Tissues and Cell Lines) using qPCR

Charlotte Dubois, Vyacheslav Lehenkyi, and Natalia Prevarskaya

9. Western-Blotting and Co-Immunoprecipitation of Endogenous STIM/ORAI and Protein Partners

Charlotte Dubois and Natalia Prevarskaya

10. Study of Endogenous CRAC Channels in Human Mast Cells using an Adenoviral Delivery System to Transduce Cells with Orai-Targeting shRNAs or with cDNAs Expressing Dominant-Negative Orai Channel Mutations

Ian Ashmole and Peter Bradding

11. Store-Operated Ca2+ Entry in Drosophila Primary Neuron Cultures

Sumita Chakraborty and Gaiti Hasan

Xuexin Zhang, Amy M. Spinelli, Wei Zhang, and Mohamed Trebak

13. Engineered Cross-Linking to Study the Pore Architecture of the CRAC Channel

Guolin Ma, Lian He, Ji Jing, Peng Tan, Yun Huang, and Yubin Zhou

14. Measurement of the CRAC Channel Fast Ca2+-Dependent Inactivation (FCDI)

Grigori Y. Rychkov

15. High Resolution Imaging of STIM/Orai Subcellular Localization using Array Confocal Laser Scanning Microscopy

Andras T. Deak, Benjamin Gottschalk, Emrah Eroglu, Rene Rost, Markus Waldeck-Weiermair, Wolfgang F. Graier, and Roland Malli

16. Single Channel Single Molecule Detection (SC-SMD) System

Laura G. Ceballos, Alexander Asanov, and Luis Vaca