Number and Spatial Distributionof IP3 Receptors UnderlyingCalcium Puffs

Jianwei Shuai, Sheila L. Dargan, Heather J. Rose and Ian Parker

Department of Neurobiology and BehaviorUniversity of California, Irvine

NIH Grant

Long Beach, Feb. 14, 2005

zx

blip

puff

Hierarchical Organization of Ca2+ Signals

Two Basic Questions:How many IP3R channels open during a puff?

How large is the width of an IP3R channel cluster?

<10 ?<300 nm ?

Averaged Trigger: Duration = 12 msec, Spatial width at half peak = 0.6 m.

Averaged Puff: Rising Time = 18 msec, Spatial width at half peak = 1.6 m, Duration at half peak = 30 msec.

Peak Ratio = Puff peak / Trigger peak = 6.7 (F/F0) / 1.1 (F/F0) = 6.1.

Time

Sp

ace

Puff Initiation with a Trigger Event

2100 2150 2200 2250 2300

8

6

4

2

0

Fluorescence

Base line

PuffRising

Trigger

F/F0

Time (msec)

Puff: Dec_06061

0 20 40 60 80 100

2

4

6

0

8

PuffRising

Trigger

Averaged Puff with Trigger Event

F/F0

Base line

Time (msec)

Possible Channel Dynamics during a Puff

Averagenumberof open

Channels

70605040302010

6

4

2

0

msec

PuffRisingTrigger

F/F0

TriggerT=12 ms

N=1

Puff RiseT=18 ms

N=? L=?N=0

Puff Model

6m

6m

6m

L : Cluster widthN : Total number of open channels during a puff

])CaEG[[EG](]Ca[]CaEG[])FlCa[[Fl](]Ca[]FlCa[

])ImCa[[Im](]Ca[]ImCa[ )0,,( ]Ca[]Ca[

T2

EET2

FF

T2

ImImCh22

Ca

2

JzyxDt

L

Free Ca2+

Immobile Buffer

EGTA

Fluo4 Dextran

Modeling a Puff

msec 12

pA 4.0

Trigger

IP3R

T

I

Free Ca2+ (M) FluoCa (M)

1000 2000 3000 4000 5000

0

20

40

60

80

100

Distance (nm)

Trigger Puff Peak

1000 2000 3000 4000 50000

5

10

15

20

25

Distance (nm)

Trigger Puff Peak

L msec 18Rising Puff T

L=760 nmN=30L=?N=?

Confocal Image of a Model Puff

0100

200300

400500

0

1000

2000

3000

4000

5000

SpatialProfile

TimeTrace

1000 2000 3000 4000 5000

0

2

4

6

8F/F

0

Distance (nm)

Trigger Puff Peak

0 20 40 60 80 100

0

2

4

6

8 F/F0

Base line

PuffRising

Trigger

Time (msec)

Averaged Puff with Trigger Event

)exp())()(

exp(

),,]([FluoCa1

),(Con

220

20

Con00

zyx

zyyxx

zyxdxdydzV

yx

1.6m

0.6

Estimating the number of open channels,and their distribution within a Cluster

Average Peak Ratio R0 = 6.0 , Average Puff Width W0=1600nm

Numberof

openchannels

duringa

puff

R=6.0, W=1600

20

20

20

20 )(1

1

)(1

1 Degree Matching

WWWRRR

Cluster Width (nm)

nm 2000nm 1200

, 5.74.5

W

R

200 400 600 800 1000

20

40

60

80

100

1.9001.9021.9041.9061.9081.9101.9121.9141.9161.9181.9201.9221.9241.9261.9281.9301.9321.9341.9361.9381.9401.9421.9441.9461.9481.9501.9521.9541.9561.9581.9601.9621.9641.9661.9681.9701.9721.9741.9761.9781.9801.9821.9841.9861.9881.9901.9921.9941.9961.9982.000

nm 2000or nm 1200

,5.7or 4.5

WW

RR

Conclusion

Width at Half Peak (nm) 600 610Trigger

Ratio

760 (400 - 1000)30 (20 - 45)

Puff

Experiment

2. Prediction

Cluster Width (nm)Open Channel No. during a Puff

<300?<10 ?

Model1. Data

160030

6.1

160032

5.9

Width at Half Peak (nm)Duration at Half Peak (ms)

Puff Peak / Trigger Peak

msec 18

msec 12

Rising Puff

Trigger

T

T

pA 4.0IP3R I

Simulation of Confocal Image

Scan line

)exp())()(

exp(),,]([FluoCa1

),(Con22

02

0

Con00

zyx

zyyxxzyxdxdydz

Vyx

[Fluo4Ca]

Channel Current

200 400 600 800 1000

20

40

60

80

pA 4.0IP3R I

pA 2.0IP3R I

pA 3.0IP3R I

pA 5.0IP3R I

pA 6.0IP3R I