Potent and specific actions of 2-Aminoethoxydiphenyl borate (2-APB) derivatives on Orai channel function

Abstract

In an effort to dissect the mechanism of SOCe activation, I used two novel 2-APB analogs (DPB162-AE and DPB163-AE) which are ~50-100 times more potent at modifying SOCe than 2-APB. In the presence of STIM1, both compounds (2 µM) differentially affected Orai subtypes, fully blocking endogenous Orai1, but not Orai2 or Orai3 mediated SOCe in DT40 Orai-specific knockout cells. Neither analog directly activated Orai3 over-expressed alone in HEK293 cells. Analysis of constitutively active Orai1 mutant, Orai1V102C, showed an increase in Ca2+ entry after application of DPB162-AE independent of STIM1. When STIM1 was co-expressed with Orai1V102C, there was no inhibitory effect of the analog on the mutant channel complex. DPB162-AE appeared to have a long term effect on the channel complex revealed a lack of SOCe 10 minutes after washout of the analog. STIM1ct-Orai1 Ca2+ entry was moderately increased by DPB162-AE yet constitutively active Stim1ct4EA-Orai1 Ca2+ entry was robustly inhibited. The activation of mutant Orai1V102C indicated the analogs are capable of interacting with Orai1, perhaps to widen the pore, and pointing to a putative mechanism of action for inhibition. FRET analysis indicated no effect on STIM1-Orai1, STIM1ct-Orai1 or SOAR-Orai1 coupling. Thus, the inhibitory effect on STIM1-Orai may be through physical alteration of Orai1 gating. Previously reported as having biphasic effect on SOCe proteins, DPB163-AE appeared to effect its potentiation exclusively via STIM2 with no evident inhibition of STIM2 SOCe. Inhibition by both analogs was mediated by STIM1. DPB162-AE and DPB163-AE had remarkable specificity on Orai1 as opposed to other Ca2+ permeant channels. Neither compound affected Ca2+ entry through TRPC3, TRPC6, or strontium entry through Cav1.2 channels at concentrations (2 µM) that completely inhibited Orai1-mediated SOCe. In summary, DPB162-AE and DPB163-AE are highly specific inhibitors of Orai1 SOCe, with little effect on Orai2 and Orai3, and no effect on other Ca2+ channels. They do not disrupt STIM-Orai coupling but may modify functional Orai1 channel structure to effect their inhibitory action on SOCe.