Automated patch-clamp technique: increased throughput in functional characterization and in pharmacological screening of small-conductance Ca2+ release-activated Ca2+ channels.
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 (I( CRAC)) 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 MOmega (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. I(CRAC) whole-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 I(CRAC) was activated by passive store depletion using EGTA. Apparent rundown of I(CRAC) was commonly observed, and the current could be reactivated by subsequent addition of thapsigargin. I(CRAC) was blocked by SKF-96365 and 2-APB with IC50 values of 4.7 +/- 1.1 microM (n = 9) and 7.5 +/- 0.7 (n = 9) microM, respectively. The potencies of these blockers were similar to values reported for I(CRAC) in 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.
Schrøder
Rikke L
RL
Sophion Bioscience A/S, Ballerup, Denmark.
Friis
Søren
S
Sunesen
Morten
M
Mathes
Chris
C
Willumsen
Niels J
NJ
D000818:Animals; D001331:Automation; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D004594:Electrophysiology / Q000379:methods; D007093:Imidazoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry; D015759:Ionomycin / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology
D007093:Imidazoles; D010975:Platelet Aggregation Inhibitors; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium