Technical Reference #1702

1702.

Imaging the quantal substructure of single IP3R channel activity during Ca2+ puffs in intact mammalian cells Ian F. Smitha;1 and Ian Parkera;b, University of California, PNAS, 106(1702), (2009)
Link To Paper

Abstract:
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.

Keywords:
calcium signaling; single-channel flux; TIRF microscopy; optical patch-clamp

Materials & Methods:
Cell Culture and Loading. Human neuroblastoma SH-SY5Y cells were cultured as previously described (17) in a mixture (1:1) of Ham’s F12 medium and Eagle MEM supplemented with 10% (vol./vol.) FCS and 1% nonessential amino acids. Cells were incubated at 37 °C in a humidified incubator gassed with95% air and 5% CO2 passaged every 7 days and used for a maximum of 20 passages. Four days before imaging cells were harvested in PBS solution without Ca2 or Mg2 and subcultured in Petri dishes with glass coverslips as the base (MatTek) at a seeding density of 3 104 cells/mL. Cells were then loaded a few hours before use by incubation with Hepes-buffered saline solution (in mM: NaCl 135; KCl 5; MgSO4 1.2; CaCl2 2.5; Hepes 5; glucose 10) containing 1 Mci-IP3/PM (SiChem) at room temperature for 45 min followed by incubation with 1 M caged ci-IP3/PM plus 5 M fluo-4 AM (Invitrogen) at room temperature for 45 min and finally 45 min with 5 M EGTA-AM (Invitrogen).

Microscopic Technique
Fluorescence Microscopy, Total Internal Reflection Fluorescence (TIRF)

Cell Type(s)
SH-SY5Y