- 1The effects of the IP3-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca2+ release-activated Ca2+ current (ICRAC) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined.
- 22-APB elicited both stimulatory and inhibitory effects on Ca2+ influx through CRAC channels. At concentrations of 1–5 μm, 2-APB enhanced Ca2+ entry in intact cells and increased ICRAC amplitude by up to fivefold. At levels ≥ 10 μm, 2-APB caused a transient enhancement of ICRAC followed by inhibition.
- 32-APB altered the kinetics of fast Ca2+-dependent inactivation of ICRAC. At concentrations of 1–5 μm, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (≥ 10 μm) reduced or completely blocked inactivation.
- 42-APB inhibited Ca2+ efflux from mitochondria.
- 52-APB inhibited ICRAC 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.
- 6Neither ICRAC activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 μg 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 ICRAC in both cell types, indicating that 2-APB exerts its effects independently of IP3 receptors.
- 8Our results show that CRAC channel activation does not require physical interaction with IP3 receptors as proposed in the conformational coupling model. Potentiation of ICRAC 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.