dsrna GFP 0 Ca 0 Ca 0 Ca TG Iono Time (s)

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Rolf Elliott
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1 Rtio (FL1/FL3) MFI dsrna GFP C dsrna dori C Rtio (FL1/FL3) MFI C 1 2 Rtio (FL1/FL3) MFI C 1 2 C 1 2 C 1 2 Supplementry Figure 1. RNAi-medited depletion of dori hs no effet on the filling stte of intrellulr C stores. S2R+ ells were inuted for 4 dys with doule-strnded (ds) RNA ginst dori (right pnels) or GFP (left pnels), loded with C inditor dyes Fluo-4 nd Fur-Red nd nlyzed for intrellulr C levels y flow ytometry. C stores were depleted in the sene of [C ] o using 1 mm thpsigrgin (), 1 mm ionomyin () or oth () t the indited time points. Note tht the relese of C from stores in ells depleted of dori is similr to tht oserved in ontrol ells (RNAi for GFP). Tres show hnges in the rtio of Fluo-4 (FL1) nd Fur Red (FL3) emission.

2 M1 L K A S S R T S A L L S G F A M V A M V E V Q L D A D Ori L K A S S R T S A L L S G F A M V A M V E V Q L D A D H. spiens NP_ L K A S S R T S A L L S G F A M V A M V E V Q L D T D M. musulus NP_78632 L K A S S R T S A L L S G F A M V A M V E V Q L D T D R. norvegius XP_ L K A S S R T S A L L S G F A M V A M V E V Q L D A D B. turus XP_ L K A S S R T S A L L S G F A M V A M V E V Q L D A D C. fmiliris XP_ L K A S S R T S A L L S G F A M V A M V E V Q L D A E G. gllus NP_25829 L K A S S R T S A L L S G F A M V A M V E V Q L E A D X. tropilis ENSXETESTP16641 L K A S S R T S A L L S G F A M V A M V E V Q L D N T T. nigroviridis CAF9927 L K A S S R T S A L L S G F A M V A M V E V Q L D T N D. rerio NP_ L K A S S K T S A L L S G F A M V A M V E V Q L D H D D. melnogster NP_ L K A S S R T S A L L A G F A M V A M V E V Q L S A T S. purpurtus XP_78791 L K A S S R T S A L L A G F A M V C L V E L Q Y D Q S C. elegns NP_ L K A S S R T S A L L S G F A M V A M V E V Q L E T Q Ori2 H. spiens L K A S S R T S A L L S G F A M V A M V E V Q L E S D Ori3 H. spiens NP_1162 NP_6891 Supplementry Figure 2. High degree of sequene onservtion in the first of four puttive trnsmemrne regions (M1, underlined) of Ori1. R91 (old) is mutted to tryptophn (W) in the SCID ptients. Ori1, FLJ14466; Ori2, C7orf19; Ori3, MGC124.

3 C C C C C C ) 8 nm6 [C 6 8 ) nm [C d Ori1 WT C C C C C 6 8 C e ) nm8 6 [C 2-APB (75 mm) 6 8 ) nm8 6 [C C C C C C f 2-APB (3 mm) 6 8 C g ) nm [C ) nm [C L Ori1 WT 6 8 ) 8 nm6 h [C L /s) nm15 Influx 5 ( Initil rte of C GFP fluoresene Supplementry Figure 3. Expression of Ori1 in firolsts from SCID ptients restores store-operted C entry. -, C influx ws ompred y single-ell video imging in firolsts from SCID ptients left untrnsdued (red tre) or trnsdued with Ori1 WT (green tre) in iistroni retrovirl vetor ontining IRES-GFP. To stimulte influx, C stores were depleted with thpsigrgin () in the sene of [C ] o followed y reddition of 2 mm C., Inhiition of C influx y 75 mm 2-APB in SCID firolsts expressing Ori1WT. d, Potentition of C influx in Ori1 WT expressing SCID firolsts y 3 mm 2-APB. e-f, Inhiition of C influx in Ori1 WT expressing SCID firolsts y 2 mm L 3+ dded efore (e) or fter (f) reddition of mm [C ] o. For eh experiment, ~ 15- GFP-positive firolsts were nlyzed; GFP-negtive SCID firolsts filed to show C influx s oserved in (dt not shown). Experiments were repeted t lest three times for eh protool. g, C influx in Ori1 WT -omplemented SCID firolsts is dependent on store depletion. C influx ws evluted y single-ell video imging. Cells were initilly inuted in C - free Ringer solution, followed y perfusion with Ringer solution ontining mm C. This protool did not result in signifint C influx, in ontrst to the store depletion nd C influx oserved when the sme inutions were repeted in the presene of thpsigrgin (). Thus Ori1 WT expression does not use store-depletion or onstitutive opening of plsm memrne C hnnels. h, Reonstitution of C influx in SCID T ells omplemented with wild-type Ori1 orreltes with GFP expression levels. Plotted is the initil rte of C influx (in nm/s) vs. GFP expression (men fluoresene intensity from -255) in SCID T ells trnsdued with Ori1 WT nd GFP, oth expressed from iistroni vetor (pmscv-cite-egfp-pgk-puro). For lultion of C influx in Ori1-omplemented SCID T ells s shown in Figure 5d, C influx ws verged in ll GFP-expressing ells (men GFP expression = 7; men C influx rte ~ 13.5 nm/s). Men fluoresene intensity ws mesured on digitl imges of GFP expression tken efore C imging using the ColorSpy tool in Openl (Improvision).

- my Merge Are of detil Ori1 WT Ori1 R91W Supplementry Figure 4.

my-tgged Ori1 WT () or Ori1 R91W () in iistroni IRES-GFP-ontining

For onfol imging, ells were fixed with 3% prformldehyde,

4 - my Merge Are of detil Ori1 WT Ori1 R91W Supplementry Figure 4. Equivlent plsm-memrne ssoited expression of wild-type nd mutnt Ori1 in SCID firolsts. Firolsts of SCID ptients were retrovirlly trnsdued with N-terminlly my-tgged Ori1 WT () or Ori1 R91W () in iistroni IRES-GFP-ontining vetor. For onfol imging, ells were fixed with 3% prformldehyde, permeilized nd stined with nti-my ntiodies. Wild-type nd mutnt Ori1 re expressed similrly t or ner the plsm memrne. Similr results were otined using C-terminlly my-tgged Ori1 WT nd Ori1 R91W.

5 ± 128 ± 32 n=3 CTRL n=5 SCID + Ori1 WT ± 1.1 n=4 CTRL 7. ± ±.8.8 ± n=4 2 n=4 n=4 SCID + CTRL SCID + Ori1 WT Ori1 WT d ms pa + mv - mv Supplementry Figure 5. Quntifition of I CRAC properties in SCID T ells reonstituted with wildtype Ori1. All dt used for quntifition were derived from experiments shown in Figure 6 for SCID T ells expressing wildtype Ori1 or from nlogous experiments on ontrol T ells (not shown)., Kinetis of urrent tivtion in ontrol T ells nd SCID T ells omplemented with wildtype Ori1, following whole-ell rek-in with 8 mm BAPTA in the pth pipette. The times for C urrents to reh hlf-mximl vlue re shown., The rtio of the pek N + urrent mesured in divlent-free (DVF) solution to the preeding C urrent in mm [C ] o, for ontrol T ells nd SCID T ells expressing wildtype Ori1., The rte of depotentition of N + urrent mesured under DVF onditions in ontrol T ells nd in SCID ells expressing wildtype Ori1. Depotentition rte is quntified s the time for the urrent to dey to hlf its pek vlue. d, Fst intivtion of the C urrent in SCID T ell expressing Ori1 WT in the presene of mm [C ] o. The extent nd time ourse of intivtion ws similr to tht previously reported for CRAC hnnels in Jurkt 38 T ells (extent of intivtion t - mv in ms:.54±5; t fst : 9±2 ms; t slow : 84±12 ms).

SUPPLEMENTARY INFORMATION. For

SUPPLEMENTARY INFORMATION. For Eletroni Supplementry Mteril (ESI) for Journl of Mterils Chemistry B. This journl is The Royl Soiety of Chemistry 217 SUPPLEMETARY IFRMATI For Phyoynin-bsed nnorrier s new nnopltform for effiient overoming