Stim-Dependent and Independent Effects of 2-APB on Orai3 Crac Channels

Abstract

The compound 2-aminoethyldiphenyl borate (2-APB) has received widespread attention for its ability to modulate store-operated CRAC channels. 2-APB elicits complex effects in native and ectopic CRAC channels arising from the over-expression of Orai1 (the pore subunit), causing a several-fold enhancement of ICRAC at low concentrations (20 μM). However, recent studies indicate that 2-APB produces strikingly different effects in the Orai3 variant. Here, high 2-APB concentrations activate (rather than inhibit) Orai3 channels. Moreover, the 2-APB activated Orai3 currents differ from store-operated Orai3 (and Orai1) currents in manifesting altered ion selectivity. The multiplicity of 2-APB effects in the different Orai isoforms has confounded efforts to understand its mode of action. Here, we find that 2-APB (50 μM) induces Orai3 current in two kinetically distinct phases: an initial increase in current with no change in ion selectivity is followed by secondary activation of Orai3 channels with altered ion selectivity. Lower concentrations of 2-APB (< 10 μM) potentiated Orai3 currents with no change in ion selectivity, resembling effects seen in Orai1. In contrast to the activation of Orai3 channels by high concentrations of 2-APB, the potentiation by low concentrations of 2-APB was entirely dependent on STIM1. High concentrations of 2-APB also eliminated fast Ca2+-dependent inactivation of Orai3 currents. Collectively, our results indicate that as seen with Orai1 and native CRAC channels, 2-APB causes dual effects on Orai3 channels: low concentrations potentiate Orai3 currents with no change in ion selectivity, whereas high concentrations activate Orai3 currents while also altering ion selectivity and removing fast inactivation. Our results suggest that the complex effects of 2-APB on Orai1 and Orai3 channels share common mechanisms.