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.

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Protein abundance in cardiomyocytes is controlled at both transcription and translation levels. While changes in transcription in cardiac proteins such as the sarcoplasmic reticulum Ca 2þ ATPAse (SERCA2a) in response to both physiological and pathological stimuli have been extensively studied, dynamic modulation of translation and the underlying mechanisms have received little attention. We have developed MR-PLISH (mRNA-rRNA Proximity Ligated in Situ Hybridization), a new imaging method for visualization of translation of endogenous specific mRNA transcripts in cardiomyocytes. This method is based on combined use of RNA in situ hybridization and the proximity ligation assay (PLA) technology and allows us to detect single mRNAs interacting with 18S ribosomal RNA (Rn18s). Using this approach, as well as RNAscopeÒ for single molecule detection of mRNA, we examined the effects of depleting intra-SR Ca 2þ with thapsigargin (TG) on transcription and translation of the mRNA for SERCA2a (Atp2a2) in cardiomyocytes. Under baseline conditions both total and 18s rRNA-hybridized Atp2a2 displayed a cell-wide distribution. Treatment with thapsigargin caused no significant changes in abundance or distribution of total Atp2a2 mRNA; however, it resulted in a significant increase in 18s rRNA-hybridized Atp2a2, consistent with an increased rate of active protein synthesis. These results suggest that intra-SR Ca 2þ modulates localized translation of SERCA2a mRNA in cardiomyocytes. Thus, SERCA2A translation maybe subject to regulation via a local feedback loop to compensate for reduction in intra-SR Ca 2þ levels.

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