The Minimal Requirements to Use Calcium Imaging to Analyze ICRAC

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 ICRAC is always preferred. Since many laboratories use Ca2+ imaging for ICRAC 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.