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 /

bas

al s

et 2

CCE / basal set 1

0 10000 20000-6

-3

0

3

6

Amplicon number

ZS

co

re o

f C

CE

/ b

asa

l

Genome-wide RNAi screen for SOC influx

1.0 1.50

250

500

1 2 30

4000

8000

# o

f am

plic

on

s

CCE / basal

Mea

n

olf

186-

FS

ec61

alp

ha

Syx

5 p vrcd

c16 CG

874

3 CG

1421

4 CG

4012

7S

yx1

A

Ca-

P60

AS

tim

Sti

m

Ave

1

2

CC

E /

ba

sa

l

hits with TMs

1

2

Top 10 hits

CC

E /

ba

sa

l

Mea

n Sti

mts

rcd

c23

olf

186-

Fd

om

Sec

61al

ph

aS

yx5

del

taC

OPS

tim

A

veEts

65A Ca-

P60

A

Genome-wide RNAi screen for SOC influx

0 500 10000

1

2

3

[C

a2+] i (

M)

Ca0 + TG

Ca0

Ca2

Time (s)0 500 1000

0

1

2

3

[Ca2+

] i (M

)

Ca0 + TG

Ca0

Ca2

Time (s)

Ca

0 +

T

G

Re

sti

n g

Ma

xim a

l

Su

sta

ine d

Ca

0

C

a2

0

1

2

3 CG11059 olf186-F

[Ca

2+] i (

M)

bp MdsRNA

CG11059

Stim olf186-F

600

500

400

Psn

600

400

300

500 Stim

CG11059200

100

300

400500

300olf186-F

200

100

BA C

D E F

0 200 400

-20

0

olf186-F

Control

I (pA

)

Time (s)

Ca2

0

4

8

12

I de

nsity

(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-P60A

400300

100

200

600

300

500400

CG11059

600500400

200

300

500600

400300

MbpdsRNA

CG11059 Stim Syx5600

300

500600

400Stim

600500400

200

300CG11059

Psn500600

400300

400500

300

200

Syx5

bp MdsRNA

CG11059 Stim tsr

600

400500

300

CG11059300

100

200

tsr200

300

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 currents

BA

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 (p

A)

Time (s)

ACa20Ca2

I (p

A)

Time (s)

a

bCa20 (b)

-100 -50 50 100V (mV)

I (pA)

-200

-400

Ca2 (a)

olf1

86-F

+st

im,

time 1/

2

olf1

86-F

+st

im,

dela

y

olf1

86-F

,

del

ay

olf1

86-F

,

tim

e 1/2

Tim

e (s

)I (p

A)

Time (s)

Delay

Time 1/2

50% of currenthas developed

olf1

86-F

+ s

tim*

olf1

86-F

stim

Con

trol

I de

nsity

(pA

/pF

)

Giant CRAC currents induced by olf186-F + Stim

BA

C D

0 150 300

-300

-150

0

CsNaI (

pA)

Time (s)

Ca2

c

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

[Ca

2+] i (

nM

)

Time (s)

Ca2

0 500 10000

1000

2000

Ca0 + TG

Ca0

[Ca2+

] i (n

M)

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 (n

M)

0

4

8

12

Cu

rre

nt

de

nsi

ty (

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

2

Ca0 + TG

Ca0

Ca2

[Ca2+

] i (M

)

Time (s)0 500 1000

0

1

2

Ca0 + TGCa2

[Ca2+

] i (M

)

Ca0

Time (s)0 500 1000

0

1

2

Ca0 + TG

Ca0

Ca2

[Ca2+

] i (M

)

Time (s)

BA

E

C

D

CG11059 Syx5 tsr

Ca

0 +

T

G

Re

sti

n g

Ma

xim a

l

Su

sta

ine d

Ca

0

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. SERCA, and Syntaxin 5: all are essential for CRAC channel function. Other hits also.Other 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 Translocation of STIM1 to the plasma membrane is induced by store depletion.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

ST

IM1

STIM1

Syntaxin5

Ca2+Ca2+

SERCA

Ca2+

IP3R

PLCγTK

DAG

PKC

IP3

PI(4,5)P2