Potentiation and inhibition of Ca2+ release-activated Ca2+ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP3 receptors

1. The effects of the IP3-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca2+ release-activated Ca2+ current (I-CRAC) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined. 2. 2-APB elicited both stimulatory and inhibitory effects on Ca2+ influx through CRAC channels. At concentrations of 1-5 mum, 2-APB enhanced Ca2+ entrv in intact cells and increased I-CRAC amplitude by up to fivefold. At levels greater than or equal to 10 mum, 2-APB caused a transient enhancement of I-CRAC followed by inhibition. 3. 2-APB altered the kinetics of fast Ca2+-dependent inactivation of I-CRAC. At concentrations of 1-5 mum, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (greater than or equal to 10 mum) reduced or completely blocked inactivation. 4. 2-APB inhibited Ca2+ efflux from mitochondria. 5. 2-APB inhibited I-CRAC more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect, potentiation or inhibition. Thus, 2-APB may have an extracellular site of action. 6. Neither I-CRAC activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 mug ml(-1) heparin. 7. I-CRAC is present in wild-type as well as mutant DT40 B cells lacking all three IP3 receptor isoforms. 2-APB also potentiates and inhibits I-CRAC in both cell types, indicating that 2-APB exerts its effects independently of IP3 receptors. 8. Our results show that CRAC channel activation does not require physical inter-action with IP3 receptors as proposed in the conformational coupling model. Potentiation of I-CRAC by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.