| Autor: |
Schrøder, Rikke L., Friis, Søren, Sunesen, Morten, Mathes, Chris, Willumsen, Niels J. |
| Zdroj: |
SLAS Discovery: Advancing Life Sciences R&D; August 2008, Vol. 13 Issue: 7 p638-647, 10p |
| Abstrakt: |
The suitability of an automated patch clamp for the characterization and pharmacological screening of calcium release–activated calcium (CRAC) channels endogenously expressed in RBL-2H3 cells was explored with the QPatch system. CRAC currents (ICRAC) are small, and thus precise recordings require high signal-to-noise ratios obtained by high seal resistances. Automated whole-cell establishment resulted in membrane resistances of 1728 ± 226 MΩ (n= 44). CRAC channels were activated by a number of methods that raise intracellular calcium concentration, including EGTA, ionomycin, Ins(1,4,5)P3, and thapsigargin. ICRACwhole-cell currents ranged from 30 to 120 pA with rise times of 40 to 150 s. An initial delay in current activation was observed in particular when ICRACwas activated by passive store depletion using EGTA. Apparent rundown of ICRACwas commonly observed, and the current could be reactivated by subsequent addition of thapsigargin. ICRACwas blocked by SKF-96365 and 2-APB with IC50values of 4.7 ± 1.1 μM (n= 9) and 7.5 ± 0.7 (n= 9) μM, respectively. The potencies of these blockers were similar to values reported for ICRACin similar conventional patch-clamp experiments. The study demonstrates that CRAC channels can be rapidly and efficiently targeted with automated patch-clamp techniques for characterization of physiological and pharmacological properties. |
| Databáze: |
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