title: Measuring endogenous ICRAC and ORAI currents with the patch-clamp technique.
abstract: 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.
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Dalia
authors.initials: D
authors.lastname: Alansary
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Kilch
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Holzmann
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Peinelt
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Hoth
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Lis
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D018408:Patch-Clamp Techniques / Q000379:methods*
chemical_list: D015220:Calcium Channels; D002413:Cations, Divalent; D002118:Calcium