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Imaging the quantal substructure of single IP3R channel activity during Ca2+ puffs in intact mammalian cells

By Ian F. Smith and Ian Parker


The spatiotemporal patterning of Ca2+ signals regulates numerous cellular functions, and is determined by the functional properties and spatial clustering of inositol trisphosphate receptor (IP3R) Ca2+ release channels in the endoplasmic reticulum membrane. However, studies at the single-channel level have been hampered because IP3Rs are inaccessible to patch-clamp recording in intact cells, and because excised organelle and bilayer reconstitution systems disrupt the Ca2+-induced Ca2+ release (CICR) process that mediates channel-channel coordination. We introduce here the use of total internal reflection fluorescence microscopy to image single-channel Ca2+ flux through individual and clustered IP3Rs in intact mammalian cells. This enables a quantal dissection of the local calcium puffs that constitute building blocks of cellular Ca2+ signals, revealing stochastic recruitment of, on average, approximately 6 active IP3Rs clustered within <500 nm. Channel openings are rapidly (≈10 ms) recruited by opening of an initial trigger channel, and a similarly rapid inhibitory process terminates puffs despite local [Ca2+] elevation that would otherwise sustain Ca2+-induced Ca2+ release indefinitely. Minimally invasive, nano-scale Ca2+ imaging provides a powerful tool for the functional study of intracellular Ca2+ release channels while maintaining the native architecture and dynamic interactions essential for discrete and selective cell signaling

Topics: Biological Sciences
Publisher: National Academy of Sciences
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Provided by: PubMed Central
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