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