Local calcium signals (‘puffs’) and their relation to
single-channel activity of IP3 receptors
Xenopus laevis and her oocyte
• Expresses only type-1 IP3R
• Imaging of signals (puffs) resulting from Ca2+ liberation through IP3R endoplasmic reticulum (Parker)
• Patch clamp recording of currents through single IP3R in nuclear membrane (Fosket/Mak)
In the presence of IP3, +ve and –ve feedback of Ca2+ on the IP3R generates repetitive, regenerative waves
Ca2+ waves in a whole cell
Time
[Ca 2+]
cyt
Comparison of puffs and sparks
Are puffs single channel events, or do they involve the concerted opening of multiple IP3R/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
How many IP3R/channels open to generate a puff ? Estimate from ‘signal mass’
Blips and puffs are seen at the same site, and blips are not small just because they are short
Calcium signals show a continuum of sizes, probably resulting from stochastic variation in both number and duration of channel openings
Imaging Ca2+ flux through single N- type voltage-gated Ca2+ channels
10 mM extracellular Ca2+ Fluo-4
Puffs are larger than single channel fluorescence signals and have greater spatial width
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
Puff kinetics and their relation to single IP3R 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
Puffs are triggered by relatively small [Ca2+] elevations
Abortive wave recruiting multiple puff sites
Puff triggered by Ca2+ pulse from laser ‘zap’
Stepwise recruitment during a puff
Puffs show ‘rectangular’ kinetics, with durations appreciable longer than mean IP3R channel open lifetime
Variability in puff durations
Dependence of puff parameters on [IP3] Problem - above a threshold [IP3] puffs sites coordinate to generate waves Solution - load EGTA (a slow Ca buffer) to uncouple puff sites
Activation of puffs at varying [IP3] after uncoupling by EGTA
Mean duration of puffs increases linearly with [IP3]
Rate of Ca2+ release during puffs increases linearly
with [IP3]
Mean duration of puffs is different with different ligands
‘Incremental’ responses of puff sites to stepwise increases in [IP3]
Hypothetical patterns of IP3R channel activity that may underlie puffs
Credits
Dr. Angelo Demuro
Dr. Jonathan Marchant
Dr. Nick Callamaras