1. Number and Spatial Distribution of IP3 Receptors Underlying Calcium Puffs Jianwei Shuai, Sheila L. Dargan, Heather J. Rose and Ian Parker Department of Neurobiology and Behavior University of California, Irvine NIH Grant Long Beach, Feb. 14, 2005

2. z x 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 ?

3. 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/F 0 ) / 1.1 (  F/F 0 ) = 6.1. Time Space Puff Initiation with a Trigger Event

4. Possible Channel Dynamics during a Puff Average number of open Channels Trigger T=12 ms N=1 Puff Rise T=18 ms N=? L=? N =0

5. Puff Model 6m6m 6m6m 6m6m L : Cluster width N : Total number of open channels during a puff L Free Ca2+ Immobile Buffer EGTA Fluo4 Dextran

6. Modeling a Puff Free Ca 2+ (  M) FluoCa (  M) L L=760 nm N=30 L=? N=?

7. Confocal Image of a Model Puff Spatial Profile Time Trace 1.6  m 0.6

8. Estimating the number of open channels, and their distribution within a Cluster Average Peak Ratio R 0 = 6.0, Average Puff Width W 0 =1600nm Number of open channels during a puff R=6.0, W=1600 Cluster Width (nm)

9. Conclusion Width at Half Peak (nm)600 610 Trigger Ratio 760 (400 - 1000) 30 (20 - 45) Puff Experiment 2. Prediction Cluster Width (nm) Open Channel No. during a Puff <300? <10 ? Model 1. Data 1600 30 6.1 1600 32 5.9 Width at Half Peak (nm) Duration at Half Peak (ms) Puff Peak / Trigger Peak

11. Simulation of Confocal Image Scan line [Fluo4Ca]

12. Channel Current