2‐Aminoethoxydiphenyl borate directly facilitates and indirectly inhibits STIM1‐dependent gating of CRAC channels

Abstract

2‐Aminoethoxydiphenyl borate (2‐APB) has emerged as a useful pharmacological tool in the study of store‐operated Ca 2+ entry (SOCE). It has been shown to potentiate store‐operated Ca 2+ release‐activated Ca 2+ (CRAC) currents at low micromolar concentrations and to inhibit them at higher concentrations. Initial experiments with the three CRAC channel subtypes CRACM1, CRACM2 and CRACM3 have indicated that they might be differentially affected by 2‐APB. We now present a thorough pharmacological profile of 2‐APB and report that it can activate CRACM3 channels in a store‐independent manner without the requirement of STIM1, whereas CRACM2 by itself is completely unresponsive to 2‐APB and CRACM1 is only very weakly activated. However, when coexpressed with STIM1 and activated via store depletion, CRACM1 and CRACM2 are facilitated at low 2‐APB concentrations and inhibited at higher concentrations, while CRACM3 only exhibits potentiated currents. Consistently, the 2‐APB‐induced CRAC currents exhibit altered selectivities that are characterized by a leftward shift in reversal potential and the emergence of large outward currents that are carried by normally impermeant monovalent cations such as Cs+ or K+. These results suggest that 2‐APB has agonistic and antagonistic modes of action on CRAC channels, acting at the channel level as a store‐independent and direct gating agonist for CRACM3 and a potentiating agonist for CRACM1 and CRACM2 following store‐operated and STIM1‐dependent activation. The inhibition of CRACM1 channels by high concentrations of 2‐APB appears to involve a direct block at the channel level and an additional uncoupling of STIM1 and CRACM1, since the compound reversed the store‐dependent multimerization of STIM1. Finally, we demonstrate that single‐point mutations of critical amino acids in the selectivity filter of the CRACM1 pore (E106D and E190A) enable 2‐APB to gate CRACM1 in a STIM1‐independent manner, suggesting that 2‐APB facilitates CRAC channels by altering the pore architecture.