1. Local calcium signals (‘puffs’) and their relation to single-channel activity of IP 3 receptors

2. Xenopus laevis and her oocyte Expresses only type-1 IP3R Imaging of signals (puffs) resulting from Ca 2+ liberation through IP 3 R endoplasmic reticulum (Parker) Patch clamp recording of currents through single IP 3 R in nuclear membrane (Fosket/Mak)

3. In the presence of IP 3, +ve and –ve feedback of Ca 2+ on the IP 3 R generates repetitive, regenerative waves Ca2+ waves in a whole cell Time [Ca 2+ ] cyt

5. Comparison of puffs and sparks

6. Are puffs single channel events, or do they involve the concerted opening of multiple IP 3 R/channels? Evidence strongly supports multi-channel origin: 1. Amount of calcium liberated is greater than can be accounted for by flux through a single channel 2. Puffs show a wide variation in amplitude 3. Fluorescence signals during puffs are greater than those associated with openings of single voltage-gated Ca2+ channels

7. How many IP 3 R/channels open to generate a puff ? Estimate from ‘signal mass’

8. Blips and puffs are seen at the same site, and blips are not small just because they are short

9. Calcium signals show a continuum of sizes, probably resulting from stochastic variation in both number and duration of channel openings

10. Imaging Ca 2+ flux through single N- type voltage- gated Ca 2+ channels 10 mM extracellular Ca 2+ Fluo-4

11. Puffs are larger than single channel fluorescence signals and have greater spatial width

12. How many channels are open during a puff ? Rate of calcium release corresponds to calcium currents of roughly 0.2 pA (blips) to 4 pA (large puffs) Calcium current through single IP3R estimated to be about 0.05 - 0.1 pA under physiological conditions (assuming 0.5 mM free [Ca2+] in e.r. lumen and including reduction of Ca conductance by permeant Mg2+ and monovalent ions) So: a large puff may involve simultaneous opening of several tens of channels

13. Puff kinetics and their relation to single IP 3 R kinetics **CICR is probably sufficient to account for recruitment of channels during a puff **Duration of calcium liberation during a puff is long in comparison to mean channel open lifetime **Puff duration varies with [IP3] and between different agonists

14. Puffs are triggered by relatively small [Ca 2+ ] elevations Abortive wave recruiting multiple puff sites Puff triggered by Ca 2+ pulse from laser ‘zap’

15. Stepwise recruitment during a puff

16. Puffs show ‘rectangular’ kinetics, with durations appreciable longer than mean IP3R channel open lifetime

17. Variability in puff durations

18. Dependence of puff parameters on [IP 3 ] Problem - above a threshold [IP3] puffs sites coordinate to generate waves Solution - load EGTA (a slow Ca buffer) to uncouple puff sites

19. Activation of puffs at varying [IP3] after uncoupling by EGTA

20. Mean duration of puffs increases linearly with [IP 3 ]

21. Rate of Ca2+ release during puffs increases linearly with [IP3]

22. Mean duration of puffs is different with different ligands

23. ‘Incremental’ responses of puff sites to stepwise increases in [IP 3 ]

25. Hypothetical patterns of IP 3 R channel activity that may underlie puffs

26. Credits Dr. Angelo Demuro Dr. Jonathan Marchant Dr. Nick Callamaras