The Minimal Requirements to Use Calcium Imaging to Analyze I_CRAC

Abstract

Endogenous calcium release-activated channel (CRAC) currents are usually quite small and not always easy to measure using the patch-clamp technique. While we have, for instance, successfully recorded very small CRAC currents in primary human effector T cells, we have not yet managed to record CRAC in naïve primary human T cells. Many groups, including ours, therefore use Ca²⁺ imaging technologies to analyze CRAC-dependent Ca²⁺ influx. However, Ca²⁺ signals are quite complex and depend on many different transporter activities; thus, it is not trivial to make quantitative statements about one single transporter, in this case CRAC channels. Therefore, a detailed patch-clamp analysis of I_CRAC is always preferred. Since many laboratories use Ca²⁺ imaging for I_CRAC analysis, we detail here the minimal requirements for reliable measurements. Ca²⁺ signals not only depend on the net Ca²⁺ influx through CRAC channels but also depend on other Ca²⁺ influx mechanisms, K⁺ channels or Cl⁻ channels (which determine the membrane potential), Ca²⁺ export mechanisms like plasma membrane Ca²⁺ ATPase (PMCA), sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) or Na⁺–Ca²⁺ exchangers, and (local) Ca²⁺ buffering often by mitochondria. In this protocol, we summarize a set of experiments that allow (quantitative) statements about CRAC channel activity using Ca²⁺ imaging experiments, including the ability to rule out Ca²⁺ signals from other sources.