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The spatiotemporal patterning of Ca(2+) signals regulates numerous cellular functions, and is determined by the functional properties and spatial clustering of inositol trisphosphate receptor (IP(3)R) Ca(2+) release channels in the endoplasmic reticulum membrane. However, studies at the single-channel level have been hampered because IP(3)Rs are inaccessible to patch-clamp recording in intact cells, and because excised organelle and bilayer reconstitution systems disrupt the Ca(2+)-induced Ca(2+) release (CICR) process that mediates channel-channel coordination. We introduce here the use of total internal reflection fluorescence microscopy to image single-channel Ca(2+) flux through individual and clustered IP(3)Rs in intact mammalian cells. This enables a quantal dissection of the local calcium puffs that constitute building blocks of cellular Ca(2+) signals, revealing stochastic recruitment of, on average, approximately 6 active IP(3)Rs clustered within &lt;500 nm. Channel openings are rapidly ( approximately 10 ms) recruited by opening of an initial trigger channel, and a similarly rapid inhibitory process terminates puffs despite local [Ca(2+)] elevation that would otherwise sustain Ca(2+)-induced Ca(2+) release indefinitely. Minimally invasive, nano-scale Ca(2+) imaging provides a powerful tool for the functional study of intracellular Ca(2+) release channels while maintaining the native architecture and dynamic interactions essential for discrete and selective cell signaling.

Imaging the quantal substructure of single IP 3 R channel activity during Ca 2+ puffs in intact mammalian cells

Imaging the quantal substructure of single IP ₃ R channel activity during Ca ²⁺ puffs in intact mammalian cells