Application Note: Recording of ICRAC in RBL-1 cells on the PatchXpress® 7000A Automated Patch Clamp System
15 October 2008
Calcium release-activated calcium (CRAC) channels play an important role in intracellular Ca2+ homeostasis. Like other store-operated calcium channels, currents through the CRAC channel (ICRAC) are activated by depletion of calcium in the endoplasmic reticulum (ER) and serve the purpose of slowly replenishing the ER with Ca2+ (Hoth and Penner, 1992; Zweifach and Lewis, 1993). Due to low densities of these channels in cells used as model systems very small currents need to be measured.
The CRAC channel is endogenously expressed in a variety of nonexcitable cells, including rat basophilic leukemia cells (RBL) and Jurkat T cells. Both RBL and Jurkat cells have been used as model systems to study the properties and regulation of the CRAC channel. Current densities in these cells are generally between 2–3 pA/pF, yielding a whole cell current in the tens of pA range. These small currents are difficult to measure and can be easily obscured by recording noise. Because of this, patch clamp recording of ICRAC requires a patch clamp system with high-performance, low-noise microelectrode amplifiers. In the PatchXpress® 7000A Automated Patch Clamp System, there are eight dual-channel highperformance micro-electrode amplifiers capable of providing low-noise measurement of small currents such as ICRAC.
To test the capability of the system, we assayed the CRAC channel in our application laboratory using a standard PatchXpress 7000A system. After optimizing the recording conditions, we successfully recorded currents from CRAC channels in RBL-1 cells. The currents were activated by either IP3, 10 mM BAPTA, or the application of thapsigargin. The recorded currents showed the characteristics of classical CRAC currents and were blocked by a number of commonly used antagonists, including SKF 96365, 2-APB and cadmium. This application note demonstrates that the PatchXpress 7000A Automated Patch Clamp System is capable of assaying CRAC channels.