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

Abstract

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 μm, 2-APB enhanced Ca²⁺ entry in intact cells and increased I_CRAC amplitude by up to fivefold. At levels ≥ 10 μm, 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 μm, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (≥ 10 μm) 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 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 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 interaction 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.