Imaging the quantal substructure of single IP_3R channel activity during Ca~(2+) puffs in intact mammalian cells

摘要

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 (IP3R) Ca~(2+) 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 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)_3Rs 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_3RS 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 [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 intraceliular Ca~(2+) release channels while maintaining the native architecture and dynamic interactions essential for discrete and selective cell signaling.