title: 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 Ca(2+) imaging technologies to analyze CRAC-dependent Ca(2+) influx. However, Ca(2+) 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 Ca(2+) imaging for ICRAC analysis, we detail here the minimal requirements for reliable measurements. Ca(2+) signals not only depend on the net Ca(2+) influx through CRAC channels but also depend on other Ca(2+) influx mechanisms, K(+) channels or Cl(-) channels (which determine the membrane potential), Ca(2+) export mechanisms like plasma membrane Ca(2+) ATPase (PMCA), sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) or Na(+)-Ca(2+) exchangers, and (local) Ca(2+) buffering often by mitochondria. In this protocol, we summarize a set of experiments that allow (quantitative) statements about CRAC channel activity using Ca(2+) imaging experiments, including the ability to rule out Ca(2+) signals from other sources.
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Dalia
authors.initials: D
authors.lastname: Alansary
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Kilch
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Holzmann
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Peinelt
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Hoth
authors.affiliation: Department of Biophysics, Saarland University, Homburg, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Lis
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D061848:Optical Imaging / Q000379:methods*
chemical_list: D015220:Calcium Channels; D002118:Calcium