Reciprocal Regulation of Store-Dependent and Independent CRAC Channel Gating by STIM1 and 2-Apb

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels generate Ca2+ signals important for gene expression, the release of inflammatory mediators, and cell motility. Under physiological conditions, CRAC channels are activated by depletion of ER Ca2+ stores via physical interactions with the ER Ca2+ sensor, STIM1. However, recent studies indicate that exogenously over-expressed Orai3 CRAC channels can also be directly activated in a store- and STIM1-independent fashion by the compound, 2-aminoethyldiphenyl borate (2-APB). Whether 2-APB also regulates store-operated gating of Orai3 channels and if STIM1 can modulate direct activation by 2-APB is unknown. Here, we report that 2-APB regulates STIM1 gating of Orai3 channels in several respects. Low concentrations potentiate Orai3 currents in a store- and STIM1-dependent manner, whereas high doses strongly inhibit store-operated Orai3 currents. Inhibition by 2-APB occurs concurrently with changes in several STIM1-dependent Orai3 channel properties, including fast inactivation and potentiation, suggesting that high doses of 2-APB affect STIM1-Orai3 coupling. Conversely, STIM1-bound Orai3 (and Orai1) channels resist direct activation by 2-APB. The resistance to 2-APB activation varies in direct proportion to 2-APB-induced inhibition of the store-operated Orai3 conductance, suggesting that 2-APB has to first uncouple STIM1 from Orai3 channels before causing direct activation. Collectively, our results indicate that the store-dependent and independent modes of CRAC channel activation are reciprocally regulated, such that channels bound to STIM1 resist 2-APB gating, while 2-APB suppresses STIM1-gating.