1. Genome-wide screen for CRAC channels

2. Treat 10 4 S2 cells 5 days with dsRNA; load with fluo-4, wash 2 Ca 2+ Ringer; 3 fluorescence measurements per well: basal, then add 1  M TG; CCE, then lyse cells; F max Calculate CCE / basal, filter out low F max x 384 wells/plate x 63 plates x 2 experimental runs for each dsRNA 145,152 fluorescence measurements 5 d in well with dsRNAFluo-4 washlyse +TG Genome-wide RNAi screen for SOC influx

3. Mean olf186-F Sec61alpha Syx5 pvr cdc16 CG8743 CG14214 CG40127 Syx1A Ca-P60A Stim Stim Ave Mean Stim tsr cdc23 olf186-F dom Sec61alpha Syx5 deltaCOP Stim Ave Ets65A Ca-P60A Genome-wide RNAi screen for SOC influx

4. Top 10 Hits DRSC amplicon target gene CCE basal basal Fmax Z of Fmax predicted TM putative function# of potential off-targets DRSC11164Ets65A1.160.23-0.350transcription factor0 DRSC04600Ca-P60A1.230.370.438SERCA pump0 DRSC20158Stim1.260.28-1.031 the putative ER Ca 2+ sensor for SOC activation DRSC04718tsr1.280.370.560actin binding protein0 DRSC02708cdc231.300.35-1.690 component of anaphase- promoting complex 1 DRSC22061olf186-F1.310.29-1.113-5 ?? 0 DRSC04558dom1.320.350.380 component of chromatin remodeling complex for DNA recombination 0 DRSC03256Sec61alpha1.320.411.4010 component of translocon complex for protein trafficking 0 DRSC03432Syx5*1.330.33-2.211 t-SNARE protein for vesicle fusion 0 DRSC18760deltaCOP1.340.32-1.390 component of COPI complex for protein trafficking 0 0

5. Ca0 + TG Resting Maximal Sustained Ca0  Ca2 BAC D EF CG11059olf186-F RNAi knock-down of olf186-F inhibits Ca 2+ influx and CRAC currents

6. Chromosome 7 12 16 olf186-F conserved gene family

7. Proposed olf186-F membrane topology Chromosome 12 homolog (ORAI1) mutated from R to W in SCID patients Feske et al., Nature in press 2006

8. Ca-P60A Stim Psn bp M dsRNA CG11059StimCa-P60A 400 300 100 200 600 300 500 400 CG11059 600 500 400 200 300 500 600 400 300 M bp dsRNA CG11059StimSyx5 600 300 500 600 400 Stim 600 500 400 200 300 CG11059 Psn 500 600 400 300 400 500 300 200 Syx5 bp M dsRNA CG11059 Stimtsr 600 400 500 300 CG11059 300 100 200 tsr 200 300 100 Psn 500 400 300 600 Stim 600 300 600 500 400 bp M 500 Stimolf186-FStim+olf186-FCont. 400 100 300 200 300 500 400 Stim Psn olf186-F RT-PCR validation of overexpression or RNAi knockdown Syntaxin 5 dsRNA tsr dsRNA SERCA dsRNA olf186-F Stim Xfection

9. Overexpression of olf186-F augments CRAC currents BA CD

10. Giant CRAC currents induced by olf186-F + Stim BA CD P Na /P Cs = 0.08Inactivation of monovalent current (divalent-free) Potentiation BlockReversible block by gadolinium

11. Classic CRAC Jurkat T Cell Lewis and Cahalan, 1989 Giant CRAC Transfected S2 Cell (olf + Stim) Zhang, Yeromin and Cahalan, 2006 Magnifying CRAC Currents From single-channel current noise estimate (7 fA at –110 mV), giant CRAC currents represent ~10 5 channels per cell, or 100 per  m 2

12. Accelerating CRAC current development by co-transfection of olf186-F with Stim

13. SERCA knockdown BA E F CD Reduced store contentElevated resting [Ca 2+ ] i and decreased SOC Decreased CRAC current

14. BA E C D CG11059Syx5tsr Ca0 + TG Resting Maximal Sustained Ca0  Ca2 Syntaxin 5 (and tsr) knockdown Decreased SOCDecreased CRAC current

15. CRAC Summary RNAi screen for SOC and single cell validation identified Stim, olf186-F, SERCA, and Syntaxin 5: all are essential for CRAC channel function. Other hits also.RNAi screen for SOC and single cell validation identified Stim, olf186-F, SERCA, and Syntaxin 5: all are essential for CRAC channel function. Other hits also. Overexpression of Stim alone does not increase CRAC current; olf186-F  3X; olf186-F + Stim  8X; faster activation kinetics.Overexpression of Stim alone does not increase CRAC current; olf186-F  3X; olf186-F + Stim  8X; faster activation kinetics. Mutation of Stim or STIM1 EF hand activates CRAC.Mutation of Stim or STIM1 EF hand activates CRAC. STIM1 is mainly in ER. STIM1 EF hand is found mainly at the plasma membrane.STIM1 is mainly in ER. STIM1 EF hand is found mainly at the plasma membrane. Translocation of STIM1 to the plasma membrane is induced by store depletion.Translocation of STIM1 to the plasma membrane is induced by store depletion. Stim may function as an ER Ca 2+ sensor that translocates to the plasma membrane to activate CRAC. olf186-F is a viable candidate for the channel. Other components? Vesicle trafficking mechanism (syntaxin 5), other?Stim may function as an ER Ca 2+ sensor that translocates to the plasma membrane to activate CRAC. olf186-F is a viable candidate for the channel. Other components? Vesicle trafficking mechanism (syntaxin 5), other?

16. Ca SOC (olf186-F) IKCa1 K+K+ TCR CaM Antigen APC MHC Ca 2+ EmEm Kv1.3 CRAC channel activation pathway? [Ca 2+ ] i STIM1 Syntaxin5 Ca 2+ SERCA Ca 2+ IP 3 R PLCγ TK DAG PKC IP 3 PI(4,5)P 2