Molecular basis of activation of the arachidonate‐regulated Ca²⁺ (ARC) channel, a store‐independent Orai channel, by plasma membrane STIM1

Key points The two known endogenous Orai channels, the calcium store‐dependent CRAC channel and the store‐independent ARC channel, are both regulated by the protein STIM1. However, whilst CRAC channel activation is regulated by STIM1 in the endoplasmic reticulum, it is the pool of STIM1 constitutively residing in the plasma membrane that regulates the ARC channels. Here we show that, although the specific regions of STIM1 critical for the regulation of these channels are largely the same, the actual mechanism of activation is markedly different. Specifically, STIM1 in the plasma membrane exists in a constitutive association with the ARC channel, only requiring arachidonic acid to induce opening of the channel. As these channels are known to play critical roles in the generation and modulation of key intracellular calcium signals, such distinct modes of activation are likely to have important implications for the generation and modulation of such signals in diverse cell types. Abstract Currently, Orai proteins are known to encode two distinct agonist‐activated, highly calcium‐selective channels: the store‐operated Ca 2+ release‐activated Ca 2+ (CRAC) channels, and the store‐independent, arachidonic acid‐activated ARC channels. Surprisingly, whilst the trigger for activation of these channels is entirely different, both depend on stromal interacting molecule 1 (STIM1). However, whilst STIM1 in the endoplasmic reticulum membrane is the critical sensor for the depletion of this calcium store that triggers CRAC channel activation, it is the pool of STIM1 constitutively resident in the plasma membrane that is essential for activation of the ARC channels. Here, using a variety of approaches, we show that the key domains within the cytosolic part of STIM1 identified as critical for the activation of CRAC channels are also key for activation of the ARC channels. However, examination of the actual steps involved in such activation reveal marked differences between these two Orai channel types. Specifically, loss of calcium from the EF‐hand of STIM1 that forms the key initiation point for activation of the CRAC channels has no effect on ARC channel activity. Secondly, in marked contrast to the dynamic and labile nature of interactions between STIM1 and the CRAC channels, STIM1 in the plasma membrane appears to be constitutively associated with the ARC channels. Finally, specific mutations in STIM1 that induce an extended, constitutively active, conformation for the CRAC channels actually prevent activation of the ARC channels by arachidonic acid. Based on these findings, we propose that the likely role of arachidonic acid lies in inducing the actual gating of the channel.