Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 106, Issue 15, Pages 6404-6409Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0810799106
Keywords
calcium signaling; single; channel flux; TIRF microscopy; optical patch-clamp
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Funding
- National Institutes of Health [GM48071]
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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 IP(3)Rs 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 IP(3)Rs 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 (approximate to 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.
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