Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca2+-selective (ARC) channels

Agonist-activated Ca2+ signals in non-excitable cells are profoundly influenced by calcium entry via both store-operated and store-independent conductances. Recent studies have demonstrated that STIM1 plays a key role in the activation of store-operated conductances including the Ca2+ -release-activated Ca2+ (CRAC) channels, and that Orail comprises the pore-forming component of these channels. We recently demonstrated that STIM1 also regulates the activity of the store-independent, arachidonic acid-regulated Ca2+ (ARC) channels, but does so in a manner entirely distinct from its regulation of the CRAC channels. This shared ability to be regulated by STIM1, together with their similar biophysical properties, suggested that these two distinct conductances may be molecularly related. Here, we report that whilst the levels of Orail alone determine the magnitude of the CRAC channel currents, both Orail and the closely related Orai3 are critical for the corresponding currents through ARC channels. Thus, in cells stably expressing STIM1, overexpression of Orail increases both CRAC and ARC channel currents. Whilst similar overexpression of Orai3 alone has no effect, ARC channel currents are specifically increased by expression of Orai3 in cells stably expressing Orail. Moreover, expression of a dominant-negative mutant Orai3, either alone or in cells expressing wild-type Orail, profoundly and specifically reduces currents through the ARC channels without affecting those through the CRAC channels, and siRNA-mediated knockdown of either Orail or Orai3 markedly inhibits ARC channel currents. Importantly, our data also show that the precise effects observed critically depend on which of the three proteins necessary for effective ARC channel activity (STIM1, Orail and Orai3) are rate limiting under the specific conditions employed.