Genome-wide screen for Genome-wide screen for CRAC channels CRAC channels

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 M TG; CCE , then lyse cells; Fmax

Calculate CCE / basal, filter out low Fmax

x 384 wells/platex 63 platesx 2 experimental runs for each dsRNA145,152 fluorescence measurements

5 d in well with dsRNA Fluo-4 wash lyse+TG

1 2 31

2

3

CC

E / b

asal

set

2

CCE / basal set 10 10000 20000

-6

-3

0

3

6

Amplicon number

Z Scor

e of C

CE

/ bas

al

Genome-wide RNAi screen for SOC influx

1.0 1.50

250

500

1 2 30

4000

8000

# of

am

plic

ons

CCE / basal

Mea

n

olf1

86-

FSe

c61a

lph

aSy

x5 p vr

cdc1

6 CG

874

3 CG

1421

4 CG

4012

7Sy

x1A

Ca-

P60ASt

im

Stim

A

ve

1

2

CC

E / b

asal

hits with TMs

1

2

Top 10 hits

CC

E / b

asal

Mea

n Sti

m tsr

cdc2

3 olf1

86-

Fdo m

Sec6

1alp

ha

Syx5

delta

CO

PStim

A

veEts6

5A Ca-

P60A

Genome-wide RNAi screen for SOC influx

0 500 10000

1

2

3

[C

a2+] i (

M)

Ca0 + TGCa0

Ca2

Time (s)0 500 1000

0

1

2

3

[Ca2+

] i (M

)

Ca0 + TGCa0

Ca2

Time (s)

Ca0

+

TG

Res

tin g

Max

im al

Sust

aine d

Ca0

C

a2

0

1

2

3 CG11059 olf186-F

[Ca2+

] i (M

)

bp MdsRNA

CG11059

Stim olf186-F

600500400

Psn

600400300

500 Stim

CG11059200100

300

400500

300olf186-F

200100

BA C

D E F

0 200 400

-20

0olf186-F

Control

I (pA

)

Time (s)

Ca2

0

4

8

12

I den

sity

(pA

/pF)

Non-treated* CG11059 olf186-F

CG11059 olf186-F

RNAi knock-down of olf186-F inhibitsCa2+ influx and CRAC currents

Chromosome 7

12

16

olf186-F conserved gene family

Proposed olf186-F membrane topology

Chromosome 12 homolog (ORAI1) mutated from R to W in SCID patients Chromosome 12 homolog (ORAI1) mutated from R to W in SCID patients Feske Feske et alet al., ., NatureNature in press 2006 in press 2006

Ca-P60A

Stim

Psn

bp MdsRNA

CG11059 Stim Ca-P60A400300

100200

600

300

500400

CG11059

600500400

200300

500600

400300

MbpdsRNA

CG11059 Stim Syx5600

300

500600

400Stim

600500400

200300

CG11059

Psn500600

400300

400500

300

200Syx5

bp MdsRNA

CG11059 Stim tsr

600

400500

300

CG11059300

100

200

tsr200300

100

Psn500400300

600

Stim

600

300

600500400

bp M500

Stim olf186-F Stim+olf186-FCont.

400

100

300200

300

500400 Stim

Psn

olf186-F

RT-PCR validation of overexpression or RNAi knockdown

Syntaxin 5dsRNA

tsrdsRNA

SERCAdsRNAolf186-F

Stim Xfection

Overexpression of olf186-F augments CRAC currentsBA

C D

0 250 500

-500

-250

0

Olf186-F + Stim

Olf186-F

Stim

ControlI (

pA)

Time (s)

Ca2

Ca20 (b)

-100 -50 50 100V (mV)

I (pA)

-200

-400

Ca2 (a)

0 250 500

-500

-250

0

0 100 200-400

-200

0

0

50

100

0 200 400

-200

-100

0

0

20

40

EED

CBCa2

olf186-F + stim

olf186-F

Stim

Control

I (pA

)

Time (s)

A Ca20Ca2

I (pA

)

Time (s)

a

bCa20 (b)

-100 -50 50 100V (mV)

I (pA)

-200

-400

Ca2 (a)

olf1

86-F

+stim

,

t

ime 1/

2

olf1

86-F

+stim

,

d

elay

olf1

86-F

,

dela

y

olf1

86-F

,

time 1/

2

Tim

e (s

)I (pA

)

Time (s)

Delay

Time 1/2

50% of currenthas developed

olf1

86-F

+ s

tim*

olf1

86-F

stim

Con

trol

I den

sity

(pA

/pF

)

Giant CRAC currents induced by olf186-F + StimBA

C D

0 150 300-300

-150

0

CsNaI (

pA)

Time (s)

Ca2c

a

b

Ca (a)

100

Na (b)

-100 -50 50V (mV)

I (pA)

-100

-200

Cs (c)

0 200 400

-200

-100

0

50 nM Gd3+

I (pA

)

Time (s)

Ca2

0 200 400

-200

-100

0

5 M 20 M 2-APB

I (pA

)

Time (s)

Ca2

PNa/PCs = 0.08

Inactivation of monovalent current

(divalent-free)

Potentiation

Block

Reversible block by gadolinium

Classic CRACJurkat T Cell

Lewis and Cahalan, 1989 Ca20 (b)

-100 -50 50 100V (mV)

I (pA)

-200

-400

Ca2 (a)

Giant CRACTransfected 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 m2

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

0 200 400

-200

-100

0

I (pA

)

Time (s)

Delay

Time 1/2

50% of currenthas developed

0 500 10000

1000

2000

Ca0 + TG

Ca0

[Ca2+

] i (nM

)

Time (s)

Ca2

0 500 10000

1000

2000

Ca0 + TG

Ca0

[Ca2+

] i (nM

)

Time (s)

Ca2

0 500 10000

200

400

[Ca2+

] i (nM

)

Time (s)

Ca2

Ca0

Ca0 + Iono

0 500 10000

200

400

[Ca2+

] i (nM

)

Time (s)

Ca2

Ca0

Ca0 + Iono

SERCA knockdown

0

1000

2000

1 2 3 4

[Ca2+

] i (nM

)

0

4

8

12

Cur

rent

den

sity

(pA

/pF)

CG11059 SERCA Pump

BA

E F

C D

Reduced store content

Elevated resting [Ca2+]i

and decreased SOC

Decreased CRAC current

0 500 10000

1

2Ca0 + TG

Ca0

Ca2

[Ca2+

] i (M

)

Time (s)0 500 1000

0

1

2Ca0 + TGCa2

[Ca2+

] i (M

)

Ca0

Time (s)0 500 1000

0

1

2Ca0 + TG

Ca0

Ca2

[Ca2+

] i (M

)

Time (s)

BA

E

C

D

CG11059 Syx5 tsr

Ca0

+

TG

Res

tin g

Max

im al

Sust

aine d

Ca0

C

a2

Syntaxin 5 (and tsr) knockdown

Decreased SOC

Decreased CRAC current

CRAC SummaryCRAC Summary• RNAi screen for SOC and single cell validation identified RNAi screen for SOC and single cell validation identified StimStim, , olf186-Folf186-F, ,

SERCA, and Syntaxin 5: all are essential for CRAC channel function. Other SERCA, and Syntaxin 5: all are essential for CRAC channel function. Other hits also.hits also.

• Overexpression of Overexpression of StimStim alone does not increase CRAC current; alone does not increase CRAC current; olf186-Folf186-F 3X; 3X; olf186-Folf186-F + + StimStim 8X; faster activation kinetics. 8X; faster activation kinetics.

• Mutation of Mutation of StimStim or STIM1 EF hand activates CRAC. or STIM1 EF hand activates CRAC.• STIM1 is mainly in ER. STIM1 EF hand is found mainly at the plasma STIM1 is mainly in ER. STIM1 EF hand is found mainly at the plasma

membrane. 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.• StimStim may function as an ER Ca may function as an ER Ca2+2+ sensor that translocates to the plasma sensor that translocates to the plasma

membrane to activate CRAC. membrane to activate CRAC. olf186-Folf186-F is a viable candidate for the channel. is a viable candidate for the channel. Other components? Vesicle trafficking mechanism (syntaxin 5), other?Other components? Vesicle trafficking mechanism (syntaxin 5), other?

CaCaSOC SOC

((olf186-Folf186-F))

IKCa1IKCa1

K+

TCR

CaM

Antigen

APC

MHC

Ca2+

Em

Kv1.3Kv1.3

CRAC channel activation pathway? CRAC channel activation pathway?

[Ca2+]i

STIM

1

STIM1

Syntaxin5

Ca2+Ca2+

SERCA

Ca2+

IP3R

PLCγTK

DAG

PKC

IP3

PI(4,5)P2