CHARACTERIZATION OF T-CELL MUTANTS WITH DEFECTS IN CAPACITATIVE CALCIUM-ENTRY - GENETIC-EVIDENCE FOR THE PHYSIOLOGICAL ROLES OF CRAC CHANNELS

Prolonged Ca2+ influx is an essential signal for the activation of T l ymphocytes by antigen. This influx is thought to occur through highly selective Ca2+ release-activated Ca2+ (CRAC) channels that are activat ed by the depletion of intracellular Ca2+ stores. We have isolated mut ants of the Jurkat human T cell line NZdipA to explore the molecular m echanisms that underlie capacitative Ca2+ entry and to allow a genetic test of the functions of CRAC channels in T cells. Five mutant cell l ines (CJ-1 through CJ-5) were selected based on their failure to expre ss a lethal diphtheria toxin A chain gene and a lacZ reporter gene dri ven by NF-AT, a Ca2+- and protein kinase C-dependent transcription fac tor. The rate of Ca2+ influx evoked by thapsigargin was reduced to var ying degrees in the mutant cells whereas the dependence of NF-AT/lacZ gene transcription on [Ca2+](i) was unaltered, suggesting that the tra nscriptional defect in these cells is caused by a reduced level of cap acitative Ca2+ entry. We examined several factors that determine the r ate of Ca2+ entry, including CRAC channel activity, K+-channel activit y, and Ca2+ clearance mechanisms. The only parameter found to be drama tically altered in most of the mutant lines was the amplitude of the C a2+ current (I-CRAC), which ranged from 1 to 41% of that seen in paren tal control cells. In each case, the severity of the I-CRAC defect was closely correlated with deficits in Ca2+ influx rate and Ca2+-depende nt gene transcription. Behavior of the mutant cells provides genetic e vidence for several roles of I-CRAC in T cells. First, mitogenic doses of ionomycin appear to elevate [Ca2+](i) primarily by activating CRAC channels. Second, I-CRAC promotes the refilling of empty Ca2+ stores. Finally, CRAC channels are solely responsible for the Ca2+ influx tha t underlies antigen-mediated T cell activation. These mutant cell line s may provide a useful system for isolating, expressing, and exploring the functions of genes involved in capacitative Ca2+ entry.