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The low-conductance, highly calciumselective channels formed by the Orai proteins are known to take two major forms, namely the storeoperated CRAC channels, and store-independent, arachidonic acid-activated ARC channels. Both are activated by STIM1, but whereas CRAC channels are activated by the stromal-interacting STIM1 located in the endoplasmic reticulum membrane, ARC channels are activated by the minor pool of STIM1 located in the plasma membrane. Critically, maximally activated CRAC channel currents and ARC channel currents are completely additive within the same cell, demonstrating that these two channels are entirely distinct entities. Moreover, the selective activation of these two Orai channels results in their ability to each induce unique distinct cellular responses. We have previously shown that the specific activation of the ARC channels requires a PKAmediated phosphorylation of a single threonine residue (T389) within the cytoplasmic region of STIM1. Examination of the molecular basis of this unique phosphorylation-dependent activation reveals that phosphorylation of the T389 residue induces a significant structural change in the key region of STIM1 that is known to interact with the Orai proteins and, specifically, it is this phosphorylation-induced structural change that determines the selective activation of the storeindependent ARC channels versus the storeoperated CRAC channels.Although originally identified as a tumor suppressor gene 1 , stromal interaction molecule 1 (STIM1) is now known to be the essential mediator of activation of the highly calciumselective Orai channels, both the store-operated CRAC channels and the store-independent ARC channels. Importantly, these channels represent key components in the activation and modulation of diverse signaling events in a multitude of different cell types. Thus, although less extensively studied, the store-independent ARC channels have been shown to play key roles in a variety of different cell types including pancreatic and parotid acinar cells 2 , pancreatic β cells 3 , and avian nasal exocrine cells 4 . The ARC channels are also present in multiple cell lines including HEK-293 cells, HeLa cells, and RBL cells 5 , as well as SY5Y neuroblastoma cells 6 and K562 erythroleukemia cells 7 . Correspondingly, STIM1 is known to be almost ubiquitously expressed in most tissues including the pancreas, skeletal muscle, brain, heart, liver and lung. Selective activation of distinct Orai channels by STIM1 2Although the molecule STIM1 is essential for the activation of both CRAC channels and ARC channels, the way in which this activation is achieved is fundamentally distinct. Thus, activation of the CRAC channels is regulated by the pool of STIM1 that is resident in the endoplasmic reticulum (ER) membrane and, physiologically, is activated on depletion of the ER calcium, typically as a result of agonistinduced activation of InsP3 receptors. This fall in ER calcium levels is sensed by STIM1 via an Nterminal calcium-binding EF-hand domain located wit...

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It has been suggested that synovial macrophages mediate nociceptive signals in knee osteoarthritis (OA) but the underlying mechanisms are unknown. Our objectives were to investigate the role of synovial macrophages and their activation via signal transducer and activator of transcription (STAT) signaling in mediating OA pain experiences. We induced experimental OA in rats via knee destabilization surgery and then performed RNA sequencing analysis in sorted synovial macrophages to identify signaling pathways associated with macrophage activation. Next, we repeated intra-articular injections of liposomal clodronate to deplete macrophages, or liposomal inhibitors of STAT1 or STAT6 to block macrophage activation, and tested the effects on local and distal mechanical pain sensitivity. We also assessed synovitis, cartilage damage, and synovial macrophage infiltration with histopathology and immunofluorescence, and crosstalk between liposomal drug-treated synovium and articular chondrocytes in co-culture. Most enriched signaling pathways in activated OA macrophages involved STAT signalling. Macrophage depletion and STAT6 inhibition led to marked, sustained improvements in mechanical pain sensitivity and synovial inflammation compared to controls, but macrophage depletion caused increased synovial fibrosis and vascularization. In contrast, STAT1 and STAT6 inhibition in macrophages did not worsen synovial or cartilage pathology. In crosstalk assays, macrophage STAT1-inhibited synovium caused the greatest increases in the expression of anabolic and catabolic chondrocyte genes and sulphated glycosaminoglycan secretion in chondrocytes. Our results suggest that synovial macrophages play a key role in mediating pain experiences in experimental knee OA, and that selectively blocking STAT6 in synovial macrophages may reduce OA-related pain without accelerating joint tissue damage.