Automated Patch-Clamp Technique: Increased Throughput in Functional Characterization and in Pharmacological Screening of Small-Conductance Ca 2+ Release-Activated Ca 2+ Channels

Abstract

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 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. 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 µM (n = 9) and 7.5 ± 0.7 (n = 9) µM, 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. (Journal of Biomolecular Screening 2008:638-647)

Keywords

automated patch clamp QPatch intracellular calcium stores CRAC channels

References

Feske S. , Gwack Y. , Prakriya M. , Srikanth S. , Puppel SH , Tanasa B. , et al: A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 2006;441:179-185.

Vig M. , Peinelt C. , Beck A. , Koomoa DL , Rabah D. , Koblan-Huberson M. , et al: CRACM1 multimers form the ion-selective pore of the CRAC channel . Curr Biol 2006;16:2073-2079.

Prakriya M. , Feske S. , Gwack Y. , Srikanth S. , Rao A. , Hogan PG : Orai1 is an essential pore subunit of the CRAC channel. Nature 2006;443:230-233.

Parekh AB : Functional consequences of activating store-operated CRAC channels . Cell Calcium 2007;42:111-121.

Mathes C. , Thompson SH : Calcium current activated by muscarinic receptors and thapsigargin in neuronal cells. J Gen Physiol 1994;104: 107-121.

Yeromin AV , Roos J. , Stauderman KA , Cahalan MD : Store-operated calcium channel in Drosophila S2 cells. J Gen Physiol 2004;123:167-182.

Estevez AY , Roberts RK , Strange K. : Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells. J Gen Physiol 2003;122(2):207-223.

Parekh AB , Putney JW : Store-operated calcium channels. Physiol Rev 2005;85:757-810.

Roos J. , DiGregorio PJ , Yeromin AV , Ohlsen K. , Lioudyno M. , Zhang S. , et al: STIM1, an essential and conserved component of store-operated Ca2+ channel function. J Cell Biol 2005;169:435-445.

10.

Partiseti M. , Le Deist F. , Hivroz C. , Fischer A. , Korn H. , Choquet D. : The calcium current activated by T cell receptor and store depletion in human lymphocytes is absent in a primary immunodeficiency . J Biol Chem 1994;269:32327-32335.

11.

Zweifach A. , Lewis RS : Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc Natl Acad Sci USA 1993;90:6295-6299.

12.

Mathes C. : QPatch: the past, present & future of automated patch clamp. Expert Opin Ther Targets 2006;10:319-327.

13.

Willumsen NJ : Increased throughput in ion channel drug development and exploration by automation of electrophysiology. Am Biotechnol Lab 2006 ;24:20-21.

14.

Mathes C. , Friis S. , Finley M. , Liu Y. : QPatch: the missing link between HTS & ion channel drug discovery . Comb Chem High Throughput Screen. In press.

15.

Bakowski D. , Glitsch MD , Parekh AB : An examination of the secretion-like coupling model for the activation of the Ca2+ release-activated Ca2+ current I(CRAC) in RBL-1 cells. J Physiol 2001;532:55-71.

16.

Bakowski D. , Parekh AB : Sarcoplasmic/endoplasmic-reticulum-Ca2+-ATPase-mediated Ca2+ reuptake, and not Ins(1,4,5)P3 receptor inactivation, prevents the activation of macroscopic Ca2+ release-activated Ca2+ current in the presence of physiological Ca2+ buffer in rat basophilic leukaemia-1 cells. Biochem J 2001;353:561-567.

17.

Prakriya M. , Lewis RS : Potentiation and inhibition of Ca2+ release-activated Ca2+ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP3 receptors. J Physiol 2001;536:3-19.

18.

Hoth M. , Penner R. : Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 1992;355:353-356.

19.

Fierro L. , Parekh AB : On the characterisation of the mechanism underlying passive activation of the Ca2+ release-activated Ca2+ current ICRAC in rat basophilic leukaemia cells. J Physiol 1999;520:407-416.

20.

Mathes C. : CRACking ion channel targets: 2nd annual ion channel targets conference . Expert Opin Ther Targets 2007;11:101-104.

21.

Hoth M. , Button DC , Lewis RS : Mitochondrial control of calcium-channel gating: a mechanism for sustained signaling and transcriptional activation in T lymphocytes. Proc Natl Acad Sci USA 2000;97:10607-10612.

22.

Prakriya M. , Lewis RS : Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels . J Gen Physiol 2002;119:487-507.

23.

Clapham DE : Sorting out MIC, TRP, and CRAC ion channels. J Gen Physiol 2002;120:217-220.