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

摘要

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.