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Genome-wide screen for CRAC channels

Genome-wide screen for CRAC channels. Genome-wide RNAi screen for SOC influx. 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 M TG; CCE , then lyse cells; F max

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Genome-wide screen for CRAC channels

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  1. Genome-wide screen for CRAC channels

  2. Genome-wide RNAi screen for SOC influx Treat 104 S2 cells 5 days with dsRNA; load with fluo-4, wash 2 Ca2+ Ringer; 3 fluorescence measurements per well: basal, then add 1 mM TG; CCE , then lyse cells; Fmax Calculate CCE / basal, filter out low Fmax x 384 wells/plate x 63 plates x 2 experimental runs for each dsRNA 145,152 fluorescence measurements 5 d in well with dsRNA Fluo-4 wash lyse +TG

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

  4. RNAi knock-down of olf186-F inhibits Ca2+ influx and CRAC currents dsRNA bp M CG11059 Stim olf186-F 500 olf186-F 400 300 200 100 600 500 Stim 400 300 300 CG11059 200 100 600 Psn 500 400 E F A B C CG11059 olf186-F D Maximal Sustained Resting Ca0  Ca2 Ca0 + TG

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

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

  7. RT-PCR validation of overexpression or RNAi knockdown dsRNA M bp CG11059 Stim Ca-P60A 400 Ca-P60A 300 200 100 600 500 Stim 400 300 600 500 400 300 CG11059 200 600 500 Psn 400 300 dsRNA dsRNA M bp Stim tsr CG11059 M bp CG11059 Stim Syx5 600 300 500 tsr 200 400 300 Syx5 100 200 600 500 Stim 600 400 500 Stim 300 400 300 300 600 CG11059 500 200 400 300 100 CG11059 200 600 500 Psn 600 Psn 400 500 400 300 300 SERCA dsRNA bp M Cont. Stim olf186-F Stim+olf186-F olf186-F Stim Xfection 500 400 300 olf186-F 200 100 600 500 Stim 400 300 600 Psn 500 400 300 tsr dsRNA Syntaxin 5 dsRNA

  8. Overexpression of olf186-F augments CRAC currents A B C D

  9. Giant CRAC currents induced by olf186-F + Stim PNa/PCs = 0.08 Inactivation of monovalent current (divalent-free) Reversible block by gadolinium Block Potentiation A B C D

  10. 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 ~105 channels per cell, or 100 per mm2

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

  12. SERCA knockdown Elevated resting [Ca2+]i and decreased SOC Reduced store content Decreased CRAC current A B C D F E

  13. Syntaxin 5 (and tsr) knockdown Decreased SOC Decreased CRAC current A B C CG11059 Syx5 tsr D E Maximal Resting Ca0  Ca2 Ca0 + TG Sustained

  14. 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. • 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. • 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. • Stim may function as an ER Ca2+ 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?

  15. PI(4,5)P2 PLCγ TK DAG IP3 PKC [Ca2+]i STIM1 Syntaxin5 STIM1 APC CRAC channel activation pathway? MHC Antigen TCR Ca2+ IP3R Kv1.3 Ca2+ Ca2+ K+ SERCA CaM IKCa1 Em CaSOC (olf186-F) Ca2+

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