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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Alansary, Dalia Kilch, Tatiana Holzmann, Christian Peinelt, Christine Hoth, Markus Lis, Annette |
| Description | Country affiliation: Germany Author Affiliation: Alansary D ( Department of Biophysics, Saarland University, Homburg, Germany.); Kilch T ( Department of Biophysics, Saarland University, Homburg, Germany.); Holzmann C ( Department of Biophysics, Saarland University, Homburg, Germany.); Peinelt C ( Department of Biophysics, Saarland University, Homburg, Germany.); Hoth M ( Department of Biophysics, Saarland University, Homburg, Germany.); Lis A ( Department of Biophysics, Saarland University, Homburg, Germany.) |
| Abstract | Although $I_{CRAC}$ and other store-operated currents are often analyzed by $Ca^{2+}$ imaging, whole-cell patch clamp, described here, is the preferred technique to analyze $I_{CRAC}$ whenever possible. The whole-cell patch-clamp protocol can even be used to record endogenous $I_{CRAC}$ in primary cells. The small endogenous current size of $I_{CRAC}$ 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 $I_{CRAC}$ currents from an overexpression system. This protocol also suggests sets of solutions that can be used for distinguishing $I_{CRAC}$ from potentially interfering currents. In addition to the solutions, the identity of $I_{CRAC}$ 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. |
| File Format | HTM / HTML |
| ISSN | 19403402 |
| Issue Number | 6 |
| Volume Number | 2014 |
| e-ISSN | 15596095 |
| Journal | Cold Spring Harbor Protocols |
| Language | English |
| Publisher | Cold Spring Harbor Laboratory Press |
| Publisher Date | 2014-06-02 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Clinical Laboratory Techniques Calcium Channels Metabolism Calcium Cations, Divalent Patch-clamp Techniques Methods Animals Cells, Cultured Humans Journal Article Research Support, Non-u.s. Gov't |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry, Genetics and Molecular Biology |
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