Patch-Clamp Measurement of I_CRAC and ORAI Channel Activity

Abstract

Depletion of internal Ca²⁺ stores activates store-operated Ca²⁺ channels. The most prominent members of this class of channels are Ca²⁺ release-activated Ca²⁺ (CRAC) channels, which are present in a variety of cell types including immune cells. CRAC channels are composed of ORAI proteins, which are activated by endoplasmic reticulum-bound STIM proteins on Ca²⁺ store depletion. The underlying Ca²⁺ current is called I_CRAC, which is required for many cellular functions including T-cell activation, mast cell activation, Ca²⁺-dependent gene expression, and refilling of internal Ca²⁺ stores. To analyze I_CRAC or the Ca²⁺ current through heterologously expressed ORAI channels, whole-cell patch clamp is the technique of choice. It allows the direct analysis of ion currents through CRAC/ORAI channels. The patch-clamp technique has been used to determine selectivity, permeability, rectification, inactivation, and several other biophysical and pharmacological properties of the channels, and is the most direct and reliable technique to analyze I_CRAC.