Measuring endogenous ICRAC and ORAI currents with the patch-clamp technique.

Area of Science:

• Cellular Physiology
• Ion Channel Research
• Biophysical Techniques

Background:

• Store-operated calcium entry (SOCE) is critical for cellular signaling.
• CRAC currents are a major component of SOCE, but their analysis can be challenging.
• Existing methods like Ca(2+) imaging have limitations for precise current analysis.

Purpose of the Study:

• To establish whole-cell patch clamp as the preferred method for analyzing ICRAC.
• To provide a detailed protocol for recording endogenous ICRAC in primary cells.
• To outline strategies for distinguishing ICRAC from other ionic currents.

Main Methods:

• Whole-cell patch clamp electrophysiology.
• Careful selection of pipette and bath solutions to minimize interference.
• Optimizing patch-clamp amplifier settings for low noise (<150 fA RMS).

Main Results:

• Demonstration of whole-cell patch clamp's suitability for endogenous ICRAC recording.
• Identification of critical parameters for successful ICRAC measurement, including solution composition and noise reduction.
• Characterization of ICRAC based on inward rectification, Ca(2+) selectivity, and reversal potential.

Conclusions:

• Whole-cell patch clamp offers superior resolution for ICRAC analysis compared to Ca(2+) imaging.
• The protocol details enable reliable recording and characterization of endogenous ICRAC.
• Understanding ICRAC properties is vital for deciphering calcium signaling pathways.