Measuring endogenous ICRAC and ORAI currents with the patch-clamp technique.

Alansary, Dalia; Kilch, Tatiana; Holzmann, Christian; Peinelt, Christine; Hoth, Markus; Lis, Annette (2014). Measuring endogenous ICRAC and ORAI currents with the patch-clamp technique. Cold Spring Harbor protocols, 2014(6), pp. 630-637. Cold Spring Harbor Laboratory 10.1101/pdb.prot073254

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Although ICRAC and other store-operated currents are often analyzed by Ca(2+) imaging, whole-cell patch clamp, described here, is the preferred technique to analyze ICRAC whenever possible. The whole-cell patch-clamp protocol can even be used to record endogenous ICRAC in primary cells. The small endogenous current size of ICRAC requires some precautions: First, it is important to inhibit potential interferences from other channels in the cell by carefully choosing the combination of pipette and bath solutions. Second, the noise should be <150 fA root mean square (RMS) when the pipette holder (with its wire) with or without a patch pipette is adjusted over (but not in!) the solution using a high amplification gain (50 mV/pA or higher) of the patch-clamp amplifier. In addition, this protocol draws attention to measures that should be considered when recording ICRAC currents from an overexpression system. This protocol also suggests sets of solutions that can be used for distinguishing ICRAC from potentially interfering currents. In addition to the solutions, the identity of ICRAC can be confirmed by the characteristic inward rectification, its high Ca(2+) selectivity, and the reversal potential of more than +50 mV. A few (mostly nonspecific) CRAC channel blockers are known, which can also be applied for characterization purposes.

Item Type:

Journal Article (Review Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Peinelt, Christine

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

1940-3402

Publisher:

Cold Spring Harbor Laboratory

Language:

English

Submitter:

Christine Peinelt

Date Deposited:

18 Jun 2018 14:24

Last Modified:

22 Oct 2019 18:28

Publisher DOI:

10.1101/pdb.prot073254

PubMed ID:

24890203

BORIS DOI:

10.7892/boris.97453

URI:

https://boris.unibe.ch/id/eprint/97453

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