S-acylation of Orai1 regulates store-operated Ca2+ entry

Abstract

Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER–PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER–PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER–PM junctions, subsequent binding to STIM1 and channel activation.

S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse

“…ORAI1 PM clusters are the macroscopic signature of ORAI1 trapping by STIM1, a dynamic event involving the entry and exit of ORAI1 particles into PM domains facing STIM1 molecules on apposed cortical ER cisternae Wu et al, 2014 . The reduced presence of ORAI1-C143A in puncta is consistent with a recent report that S-acylation is required for the recruitment of active ORAI1 channels to STIM1 clusters West et al, 2022 . Reduced ORAI1 trapping by STIM1 would reduce CRAC currents and possibly FCDI by increasing the amount of STIM1 molecules required to trap and gate deacylated channels.…”

“…A comparable inhibition was observed with cysteine-less ORAI1 in an earlier study focusing on Cys195 substitutions that prevent I CRAC inhibition by hydrogen peroxide Bogeski et al, 2010 . These data indicate that the ORAI1 channel is S-acylated at Cys143 and that replacement of this residue, but not of the two other ORAI1 cysteines, prevents S-acylation and impacts channel function, consistent with the recent report that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca 2+ store depletion West et al, 2022 . Cys143 is the only cysteine conserved in all human isoforms and in ORAI1 homologs up to C. elegans , suggesting that S-acylation at this site is an evolutionary conserved function.…”

Dynamic S-acylation of STIM1 is required for store-operated Ca2+ entry

“…This hypothesis is supported by our findings that both Orai1 and STIM1 reach peak S-acylation levels at the same time point after T cell stimulation (Fig. 1B-C; (11)). Similarly, we have observed in TIRF time lapse imaging experiments that the peak colocalization of Orai1 and STIM1 happens approximately at the same time after store depletion (Fig.…”

“…STIM1 can bind phosphatidylinositol 4,5-bisphosphate (PIP 2 ) via a lysine-rich C-terminal domain, and studies have shown that STIM1 translocates and binds PIP 2 present specifically in lipid rafts (10). This indicates an active mechanism for targeting STIM1 to lipid rafts, as we and others have shown previously for Orai1 (11,12). Perhaps not surprising, it has also been shown that there is reduced mobility of Orai1 and STIM1 after store depletion at ER-PM contact sites (13,14).…”

“…In contrast to other post-translational lipid modifications like prenylation, myristoylation, or others, S-acylation is reversible and highly labile (15). We have recently shown that Orai1 is dynamically S-acylated after store depletion and during initial stages of T cell activation (11). This was required for Orai1 activation and recruitment into puncta.…”