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

Efficient immune responses require Ca2+ fluxes across ORAI1 channels during engagement of T cell receptors (TCR) at the immune synapse (IS) between T cells and antigen presenting cells. Here, we show that ZDHHC20-mediated S-acylation of the ORAI1 channel at residue Cys143 is required for TCR assembly and signaling at the IS. Cys143 mutations reduced ORAI1 currents and store-operated Ca2+ entry in HEK-293 cells and nearly abrogated long-lasting Ca2+ elevations, NFATC1 translocation, and IL-2 secretion evoked by TCR engagement in Jurkat T cells. The acylation-deficient channel had reduced mobility in lipids, accumulated in cholesterol-poor domains, formed tiny clusters, failed to reach the IS and unexpectedly also prevented TCR recruitment to the IS. Our results establish S-acylation as a critical regulator of ORAI1 channel assembly and function at the IS and reveal that local Ca2+ fluxes are required for TCR recruitment to the synapse.


Introduction 13
The development of an efficient immune responses by T lymphocytes require long-lasting Ca 2+ 14 elevations mediated by the plasma membrane (PM) channel ORAI1 during engagement of T cell 15 receptors (TCR) at the immune synapse (IS) forming between T cells and antigen-presenting cells. 16 Following TCR engagement, the Ca 2+ depletion of the endoplasmic reticulum (ER) causes the ER-bound 17 Ca 2+ sensors STIM1-2 to oligomerize and to accumulate in ER-PM junctions, where they trap and gate 18 the Ca 2+ -release-activated (CRAC) ORAI1 channel. The ensuing Ca 2+ influx sustains long-lasting Ca 2+ 19 signals that initiate gene expression programs of T cell proliferation and differentiation. Proper ORAI1 20 function is essential for immunity in humans and patients with ORAI1 mutations suffer from severe 21 combined immunodeficiency 1 . Recent studies have revealed the structural rearrangements occurring 22 within ORAI1 as STIM1 binding opens the channel pore and increases its selectivity for Ca 2+ 2, 3 , 23 reviewed in 4 . Crystal structure from the highly homologous Drosophila Orai1 channel revealed a 24 hexamer of four concentric TM subunits, with pore-lining TM1 helixes bearing an acidic selectivity 25 filter followed by hydrophobic and basic regions 5, 6 . The closed structure is stabilized by multiple 26 interactions between interlocking TM2 and TM3 helixes and peripheral TM4 helixes, bent in three 27 crossed helical pairs extending in the cytosol. STIM1 binds to the external M4 helix, generating a gating 28 signal transmitted by the TM2/TM3 ring to TM1, opening the channel pore and increasing its Ca 2+ 29 selectivity. The reversible switch of ORAI1 between a quiescent to an active state is highly regulated 30 to avoid inappropriate Ca 2+ fluxes at the wrong time or place (reviewed in 4 ). 31

Results 59
The ORAI1 channel can undergo S-acylation on Cysteine 143. 60 Orai1 can potentially be S-acylated according to the SwissPalm 2.0 S-acylation database 61 (https://swisspalm.org/) that compiles palmitoyl-proteomes 18 . To validate that ORAI1 can undergo S-62 acylation, we assessed whether PEG-5k or tritiated palmitate could be incorporated by palmitoyl-63 thioester bonds into endogenous ORAI1 channels. HeLa cells were lysed in the presence of N-64 ethylmaleimide (NEM) to block free thiols, treated or not with hydroxylamine (HA) to break acyl-65 thioester bonds, and then with PEG-5k to label S-acylation sites. A mobility shift was observed in the 66 presence of PEG-5k on western blots with anti-ORAI1 antibodies (Fig. 1A). Tritiated palmitate was 67 detected by autoradiography in HeLa cells labelled for 2 h with 3 H-palmitic acid and 68 immunoprecipitated with anti-ORAI1 antibodies (Fig. 1B). These data show that endogenous ORAI1 69 channels incorporate palmitic acid and can be labelled by acyl exchange of the acyl moiety with PEG-70 5k. A single band of higher molecular weight was observed in the acyl-PEG assay, indicating that a 71 single residue of the ORAI1 channel can be S-acylated in these conditions. The fact that a non-shifted 72 ORAI1 band remains indicates that only a sub-population undergoes S-acylation under our 73 experimental conditions. 74 S-Acylation occurs on cysteine residues, present in ORAI1 at positions 126, 143 and 195, with 75 C143 conserved up to C.elegans (Fig. S1A) and C195 facing the extracellular side (Fig. S1B). To test 76 whether C126 and/or C143 are S-acylation sites we overexpressed ORAI1-GFP fusion proteins bearing 77 substitutions at these residues in HeLa cells and evaluated 3 H-palmitate incorporation by 78 autoradiography. Cells expressing ORAI1-GFP bearing the C143A substitution or the double 79 C126A/C143A mutation, but not the single C126A mutation, failed to incorporate 3 H-palmitate (Fig.  80 1C). Identical results were obtained with these ORAI1-GFP mutants expressed in RPE1 cells (Fig. 1D), 81 establishing that ORAI1 channels can undergo palmitoylation at residue C143. 82 S-acylation can alter ion channel trafficking, gating, and distribution in membrane lipids. To 84 understand if S-acylation could affect ORAI1 activity we measured Ca 2+ fluxes carried by ORAI1-GFP 85 fusion constructs bearing substitutions at C126 and C143. In HEK-293 cells lacking all three ORAI 86 isoforms (HEK-TKO, kindly provided by Rajesh Bhardwaj 17 ), expression of wild-type ORAI1 87 reconstituted Ca 2+ fluxes upon store depletion (Fig. 1E-F). C143 substitutions by alanine or serine, but 88 not C126 substitutions, reduced ORAI1-mediated SOCE (Fig. 1E-F). These findings were confirmed by 89 alanine substitutions in HEK-293 cells stably expressing mCherry-STIM1 (mCh-STIM1) and ORAI1-GFP, 90 (HEK-S1/O1). Although these cell lines were sorted for the same fluorescence and presented 91 comparable STIM1 and ORAI1 levels as judged by epifluorescence microscopy (Fig. S1C), SOCE 92 responses were strongly reduced in cells bearing the C143A mutation ( Fig. 2A-B and S1D). We then 93 recorded ICRAC currents in HEK-S1/O1-WT and -C143A cell lines and observed a current density 94 reduction of 5-fold in cells expressing the C143A mutant ( Fig. 2C-D). The currents retained the inward 95 rectification, positive reversal potential, and Gd 3+ -sensitivity characteristic of highly Ca 2+ selective 96 CRAC currents ( Fig. 2C and 2E) but activated more slowly and failed to inactivate in a significant 97 fraction of C143A cells (Fig 2F and S2). Our Ca 2+ imaging and electrophysiological data thus establish 98 that replacing the S-acylated Cys 143 residue within ORAI1 reduces the CRAC channel function. 99 Acylation increases ORAI1 cluster size, PM mobility, and affinity for lipid rafts. 100 To gain insight into the underlying mechanism, we then recorded the formation of STIM1 and 101 ORAI1 clusters during store depletion by TIRF microscopy. ORAI1-C143A clusters were tinier and 102 occupied a smaller fraction of the TIRF plane ( Fig. 3A-B). In contrast, the morphometric parameters of 103 mCh-STIM1 clusters were not altered (Fig. S3). Lipid incorporation into proteins changes their 104 lipophilic preference, and potentially their membrane mobility. To assess ORAI1 mobility in the PM 105 we used fluorescence recovery after photobleaching (FRAP) and measured the lateral diffusion of 106 ORAI1-GFP in HEK-S1/O1-WT and -C143A cell lines. The C143A mutant had a significantly lower 107 diffusion coefficient indicative of a reduced mobility in membrane lipids (Fig. 3C). The addition of an 108 acyl chain to transmembrane proteins increases their hydrophobicity, which may promote their 109 experiments indicate that PAT20 mediates ORAI1 S-acylation and that this post-translational 136 modification enhances SOCE. 137

ORAI1 S-acylation is required for TCR-mediated long-lasting Ca 2+ elevations in Jurkat T cells 138
Orai1 activity is critical for the function of B and T lymphocytes, which fittingly express PAT20 139 but neither PAT3 nor PAT7 (Fig. S5A, http://www.humanproteomemap.org). To assess whether ORAI1 140 S-acylation by PAT20 impacts T cell function, we generated by CRISPR an ORAI1-deficient Jurkat T cell 141 line, in which SOCE was severely blunted (Fig. 5A and S5B-C). Stable transduction of ORAI1-WT 142 restored SOCE in these cells while ORAI1-C143A expressed at comparable levels was less effective 143 ( Fig. 5A and S5D). Activation of the T cell receptor (TCR) with CD3/CD28 beads evoked long-lasting Ca 2+ 144 elevations in CRISPR ORAI1 + ORAI1-WT stable cells (Fig. 5B). In contrast, cells reconstituted with 145 ORAI1-C143A exhibited delayed responses of much smaller amplitude and duration upon TCR 146 engagement. The Ca 2+ signalling defect persisted when these cells were stimulated with TCR-coated 147 beads in Ca 2+ -free medium, and subsequent Ca 2+ readmission evoked minimal Ca 2+ responses ( Fig 5C). 148 This indicates that the physiological Ca 2+ signals engaged by the TCR receptors are severely affected 149 in Jurkat cells expressing acylation-deficient ORAI1-C143A. Accordingly, PAT20 expression augmented 150 SOCE in WT cells but had no effect in cells lacking ORAI1 (Fig. S5F). We then tested whether the 151 downstream responses of T cells were similarly affected. Cells bearing the C143A mutation had 152 reduced nuclear translocation of the transcription factor NFATC1 and IL-2 production following 153 stimulation with Tg or CD3 beads ( Fig. 5D-F-E and S5E). Furthermore, ORAI1 ablation prevented the 154 potentiating effects of PAT20 expression on the IL-2 secretion evoked by Tg and CD3/CD28 beads (Fig.  155   S5G). These results indicate that ORAI1 S-acylation at C143A is required for efficient activation of 156 Jurkat T cells following TCR engagement. 157 ORAI1 S-acylation sustains TCR assembly and signaling at the immune synapse 158 TCR activation triggers the formation of an immune synapse (IS) between T cells and antigen-159 presenting cells, a structure that maximizes the membrane contact area and organizes TCR and 160 signalling proteins into concentric zones 20 . ORAI1 channels are rapidly recruited into the IS 21, 22 and 161 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 4, 2021. ; https://doi.org/10.1101/2021.02.03.429577 doi: bioRxiv preprint are required for the formation of dynamic actin structures 23 in a self-organizing process enabling long-162 lasting local Ca 2+ signals to initiate gene expression programs that drive T cell proliferation 24 . To test 163 whether S-acylation impacts the recruitment of ORAI1 to the IS, we imaged CRISPR mediated ORAI1 164 deficient cells reconstituted with WT or mutant ORAI1-GFP during stimulation with antigen-coated 165 beads or during plating on coverslips coated with anti-CD3 mAb 25 . As previously reported, ORAI1-GFP 166 accumulated at sites of bead contact, decorating dynamic cup structures labelled with SiR-Actin (Fig.  167   6A). Fewer SiR-Actin cups were observed in CRISPR ORAI1 Jurkat cells reconstituted with ORAI1-C143A, 168 and the mutated GFP-tagged channel was not enriched at sites of contact when cups were detected 169 ( Fig. 6B). To better visualize the molecular organization of the IS, we performed TIRF imaging in 170 coverslips coated with anti-CD3 mAb. ORAI1-GFP accumulated into contact zones surrounded by SiR-171 Actin rings. The formation of actin rings was severely compromised in cells reconstituted with the S-172 acylation-deficient ORAI1-C143A channel, which failed to accumulate at contact sites ( Fig. 6C and S6A 173 Suppl Video 1 and 2). The few rings forming in C143A mutant expressing cells had a comparable actin 174 area ( Fig. S6B) but contained less ORAI1-associated GFP fluorescence, detected predominantly in the 175 centre of the IS ( Fig. 6D and S6C). Unexpectedly, TCR cluster formation was also severely impaired in 176 CRISPR ORAI1 cells reconstituted with acylation-deficient ORAI1. Although the two cell lines had 177 comparable TCR surface expression (Fig. S6D), the number of CD3 immunoreactive dots within the IS 178 was reduced 3-fold while their intra-IS distribution remained unaltered in cells reconstituted with 179 ORAI1-C143A ( Fig. 6E and S6C). The extent of co-localisation between ORAI-GFP and CD3 180 immunoreactivity was reduced in these cells, confirming the differential distribution of the two 181 proteins in the IS ( Fig. 6F and S6D). These data indicate that S-acylation is required for the recruitment 182 of the ORAI1 channel to the IS and for the formation of TCR clusters that determine the intensity and 183 duration of TCR signalling at the synapse (Fig. 6G). 184 185 186 187 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in In this study, we show that S-acylation of ORAI1 at a single cysteine residue enhances the 189 affinity of the channel for cholesterol rich lipid microdomains and promotes its trapping at the immune 190 synapse, thereby enabling the local Ca 2+ fluxes that control the proliferation of T cells. Using acyl-PEG 191 exchange, palmitate incorporation, and mutagenesis, we show that ORAI1 can be chemically modified 192 by S-acylation and identify the acylation site as Cys143 on the cytosolic rim of the second TM domain. 193 Substitutions at Cys143 but not at Cys126 within TM2 prevented palmitate incorporation and 194 decreased SOCE as well as ICRAC. A comparable inhibition was observed with cysteine-less ORAI1 in an 195 earlier study focusing on Cys195 substitutions that prevent ICRAC inhibition by hydrogen peroxide 16 . 196 These data indicate that the ORAI1 channel is S-acylated at Cys143 and that replacement of this 197 residue, but not of the two other ORAI1 cysteines, prevents S-acylation and impacts channel function. 198 Cys143 is the only cysteine conserved in all human isoforms and in ORAI1 homologs up to C.elegans, 199 suggesting that S-acylation at this site is an evolutionary conserved function. 200 Using Ca 2+ imaging and electrophysiology, we establish that ORAI1 S-acylation has a significant 201 functional impact on the channel function. Substitutions at Cys143, but not at Cys126, decreased SOCE 202 by 50% in HEK-293 cells when the channel was transiently expressed alone and by 80% when it was 203 stably co-expressed with STIM1. SOCE was also reduced when the S-acylation-defective ORAI1-C143A 204 was expressed in HEK-293 cells lacking all ORAI isoforms or in Jurkat T cells lacking ORAI1, firmly linking 205 the SOCE defect to the ORAI1 Cys143 mutation. Patch-clamp recordings confirmed that ICRAC currents 206 were reduced by 80% by the mutation when ORAI1-GFP was stably expressed together with mCh-207 STIM1, at identical expression levels. ORAI1-C143A currents retained the characteristic inward 208 rectification and high Ca 2+ selectivity of CRAC channels but activated more slowly and failed to 209 inactivate in a significant fraction of cells perfused with 10 mM BATPA in the pipette solution. This 210 indicates that the C143A mutation does not grossly alter the gating or permeation properties of the 211 CRAC channel. Instead, its main effect is to decrease the amplitude and to delay the activation of CRAC 212 currents. 213 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in We further identify the zinc-finger and DHHC-containing S-acyltransferase zDHHC20 (PAT20) 214 as mediating the S-acylation reaction. Among an array of PAT exogenously expressed in HeLa cells, 215 PAT20 was the only isoform that increased the incorporation of tritiated palmitate into full-length 216 ORAI1-GFP (Orai1). PAT3 and PAT7 promoted palmitate incorporation in a lower band corresponding 217 to a shorter form of ORAI1 (Orai1). PAT20 co-localized with ORAI1-GFP at the cell cortex and unlike 218 PAT3 and PAT 7 promoted SOCE when expressed. S-acylation by PAT20 thus positively modulates the 219 activity of both endogenous and exogenously expressed ORAI1 channels. Importantly, SOCE 220 potentiation was not observed when PAT20 was co-expressed with the acylation-deficient ORAI1-221 C143A mutant. This indicates that Cys143 is required for the potentiation by PAT20. Since ORAI1-222 C143A was co-expressed with STIM1 in these experiments, they also indicate that potential S-acylation 223 sites on STIM1 are not relevant for the effect of PAT20. These data indicate that PAT20-mediated S-224 acylation at Cys143 enhances ORAI1 channel function. 225 Using biochemical and imaging approaches, we then show that mutating the Cys143 S-226 acylation site reduces the size of ORAI1 PM clusters during SOCE. We further show that the mutation 227 reduces ORAI1 mobility in the PM and prevents its accumulation in ordered lipid domains rich in 228 cholesterol. ORAI1 PM clusters are the macroscopic signature of ORAI1 trapping by STIM1, a dynamic 229 event involving the entry and exit of ORAI1 particles into PM domains facing STIM1 molecules on 230 apposed cortical ER cisternae 26 . Molecularly, ORAI1 trapping reflects the interactions between the 231 STIM1 CAD domain and ORAI1 C terminal tail, with residues within ORAI1 M4 helix being critical for 232 trapping and gating. STIM1 clusters, on the other hand, reflect interactions between its lysin-rich C 233 terminal tail and PM domains rich in negatively charged phospholipids such as PIP2. STIM1 therefore 234 traps ORAI1 in PIP2-rich domains, while S-acylation increases ORAI1 affinity for cholesterol-rich 235 domains. The increased mobility of S-acylated ORAI1 in cholesterol-rich domains likely increases its 236 trapping by STIM1 into neighboring PIP2-rich domains since the escape probability of ORAI1 from 237 STIM1-ORAI1 complexes is <1% 26 . Increased ORAI1 retention at ER-PM contact sites would promote 238 the formation of larger channel clusters and enhance transmembrane Ca 2+ fluxes while preserving the 239 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 4, 2021. ; https://doi.org/10.1101/2021.02.03.429577 doi: bioRxiv preprint biophysical properties of the channel, consistent with our observations. Alternatively, the formation 240 of large clusters could reflect an increased affinity of S-acylated ORAI1 for STIM1 or increased lateral 241 interactions between S-acylated channels leading to the formation of high-order channel multimers 242 corresponding to the larger clusters. 243 By re-expressing the acylation-resistant ORAI1-C143A in ORAI1-deficient Jurkat T cells, we 244 show that ORAI1 S-acylation is required for the efficient activation of T lymphocytes during TCR 245 engagement. Replacing the single ORAI1 S-acylation site strongly reduced the long-lasting Ca 2+ 246 elevations driven by TCR engagement and the ensuing NFATC1 translocation and IL-2 production, 247 signature markers of T cell activation. Unexpectedly, the responses were also reduced in Ca 2+ -free 248 conditions, indicating that the inhibition of TCR signalling does not simply reflect the impaired channel 249 function of ORAI1-C143A at the cell surface. Expressing PAT20 increased Ca 2+ responses and TCR-250 induced IL-2 secretion in WT but not in ORAI1-deficient Jurkat T cells, confirming that ORAI1 S-251 acylation positively modulates TCR signalling. The reliance on S-acylation was most apparent at the IS, 252 the specialized membrane contact area that form at the interface between T cells and an antigen 253 presenting cells (APC). We observed three major synapse assembly defects in Jurkat T cells 254 reconstituted with ORAI1-C143A. First, fewer synapses formed in ORAI1-C143A exposed to CD3-255 coated beads or plated on activating coverslips. Second, ORAI1-C143A was poorly recruited to the IS 256 and the mutant channels accumulated in the IS centre. The IS contains a high percentage of highly 257 ordered lipids 27 forming lipid rafts migrating to its periphery 28 . S-acylation might target ORAI1 258 channels to these cholesterol-rich regions to optimize Ca 2+ signalling efficiency at the synapse 259 periphery (Fig. 6G). Third, the formation of TCR clusters was strongly reduced by the lack of ORAI1 S-260 acylation. This defect was unexpected as ORAI1 was not previously reported to control the molecular 261 dynamics of TCR. During strong antigenic stimuli, TCR form clusters with associated scaffolding and 262 signalling proteins that segregate in three concentric zones of the IS 29, 30 . The clusters migrate from 263 the periphery towards the centre of the IS where they are sorted for degradation 31, 32 , the strength of 264 signalling reflecting a balance between the formation of new clusters in the periphery and their 265 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 4, 2021. ; https://doi.org/10.1101/2021.02.03.429577 doi: bioRxiv preprint disassembly in the centre 33 . Defective ORAI1 targeting might impact TCR dynamics in several ways. In 266 quiescent T cells, Ca 2+ fluxes across ORAI1 channels might disrupt the CD3-lipid interactions that 267 prevent spontaneous TCR phosphorylation 34 , enhancing the activity state of TCR prior to their 268 engagement. ORAI1 targeting to specialized PM domains such as filopodia might be required for this 269 priming effect to occur. Alternatively, ORAI1 channels might control the rates of TCR recycling via 270 endosomes by promoting the activity of Ca 2+ -dependent actin-severing proteins such as gelsolin. Our 271 unexpected observation that ORAI1-C143A hinders TCR signalling even in the absence of extracellular 272 Ca 2+ suggests that ORAI1 might act from an intracellular location to promote endosomal recycling. The 273 presence of ORAI1 channels in endosomes is well documented 35 , but whether these channels mediate 274 Ca 2+ efflux from endosomes is not known. Preventing ORAI1 targeting could also impact the location 275 or activity of integrin receptors such as ICAM-1, thereby indirectly altering the formation of TCR 276 clusters. Further experiments are required to establish whether ORAI1 S-acylation promotes its 277 endocytosis and whether S-acylation is dynamic or a one-off event that can impact the affinity of 278 ORAI1 for accessory proteins or its potential interactions with other channels such as TRPC. 279 In summary, our findings reveal that the ORAI1 channel is regulated by S-acylation. The fatty 280 acid addition is mediated by PAT20 and targets the channel to lipid-ordered PM domains rich in 281 cholesterol, thereby facilitating channel trapping by STIM1 during cellular activation. The acylation-282 deficient channel failed to accumulate in the IS and prevented the formation of TCR clusters during 283 TCR engagement, severely impeding the signals that drive T cell proliferation. We propose that S-284 acylation dynamically targets the ORAI1 channel to peripheral regions of the synapses rich in 285 cholesterol to ensure efficient T cell signalling following TCR engagement. or C1267C143A were first infected with Cherry STIM1 p2K7 lentiviral vector, sorted, and then infected 317 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 4, 2021. ; https://doi.org/10.1101/2021.02.03.429577 doi: bioRxiv preprint for the indicated mutants at a MOI of 2 and sorted for the same Cherry-STIM1 and ORAI1-GFP 318 intensity. Indicated constructs were subcloned into a pWPT vector and co transfected with pCAG-319 VSVG/psPAX2 into HEK-293T cells to produce viral particles as described in 36 . Briefly, indicated 320 constructs were subcloned into a pWPT vector and co transfected with pCAG-VSVG/psPAX2 into HEK-321 293T cells to produce viral particles. After accumulation, ultracentrifugation and titration of the virus 322 these were stored at -80°C. Jurkat T clone E6 cells were purchased from ECACC and grown in RPMI . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

Radiolabeling 3H-palmitic acid incorporation 344
To follow S-acylation, transfected or non-transfected cells were incubated 1 hour in medium without 345 serum (Glasgow minimal essential medium buffered with 10 mM Hepes, pH 7.4), followed by 2 hours 346 at 37°C in IM with 200 µCI /ml 3H palmitic acid (9,10-3H(N)), washed with cold PBS prior 347 immunoprecipitation overnight with anti-ORAI antibodies and protein G-beads or anti-GFP agarose-348 coupled beads. Beads were incubated 5 minutes at 90°C in reducing sample buffer prior to SDS-PAGE. 349 Immunoprecipitates were split into two, run on 4-20% gels and analysed either by autoradiography 350 (3H-palmitate) after fixation (25% isopropanol, 65% H2O, 10% acetic acid), gels were incubated 30 351 minutes in enhancer Amplify NAMP100, and dried; or Western blotting. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in  FRAP was performed in HEK-293 S1/O1 under resting condition using the same microscope. ORAI1 400 FRAP was accomplished by following the protocol previously described 39 . Briefly, we used a live 401 chamber at 37°C and 5% CO2. Pinhole was settled at 1AU and images were sampled every 3 seconds 402 for 100 images. Bleaching was for 20 seconds (488nm 100% output) after 1 minute of basal acquisition. 403 ROI of interest was compared to the same size ROI in the same field of view and normalized to basal. 404 Traces were fitted with an exponential one-phase association model to obtain the half-life, τ1/2 and 405 fluorescence recovery. Diffusion coefficient was calculated with the formula D = 0.224r 2 /(τ1/2), in 406 which r is the radius of the bleached circle region as described in 39 . 407 with Hoesch 1:5000 for 1h at RT. For NFAT translocation Jurkat cells were treated with the indicated 449 compounds (Tg 1µM or CD3 plastic coated plates) for the indicated times and seeded into poly-L lysine 450 coated coverslips for 15 minutes at RT. Immunofluorescences for NFATC1 were performed as 451 described for HeLa cells. NFATC1 analysis was done by dividing the nuclear to the cytosolic (total-452 nuclear) pixel intensity per cell into 3 to 5 randomized fields per condition. Images were obtained in 453 a LSM700 Nikon microscope. 454

Image analysis and statistics 455
All images were analysed using ImageJ software. Co-localisation and particle concentric counting for 456 TCR were performed by applying a previously described macro 42 . 457

Data availability 458
The data that support the findings of this study are available from the corresponding author upon 459 reasonable request. 460 474 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in  . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in