Electroporation loading and flash photolysis to investigate intra- and intercellular Ca2+ signaling.
Many cellular functions are driven by variations in the intracellular Ca(2+) concentration ([Ca(2+)]i), which may appear as a single-event transient [Ca(2+)]i elevation, repetitive [Ca(2+)]i increases known as Ca(2+) oscillations, or [Ca(2+)]i increases propagating in the cytoplasm as Ca(2+) waves. Additionally, [Ca(2+)]i changes can be communicated between cells as intercellular Ca(2+) waves (ICWs). ICWs are mediated by two possible mechanisms acting in parallel: one involving gap junctions that form channels directly linking the cytoplasm of adjacent cells and one involving a paracrine messenger, in most cases ATP, that is released into the extracellular space, leading to [Ca(2+)]i changes in neighboring cells. The intracellular messenger inositol 1,4,5-trisphosphate (IP3) that triggers Ca(2+) release from Ca(2+) stores is crucial in these two ICW propagation scenarios, and is also a potent trigger to initiate ICWs. Loading inactive, "caged" IP3 into cells followed by photolytic "uncaging" with UV light, thereby liberating IP3, is a well-established method to trigger [Ca(2+)]i changes in single cells that is also effective in initiating ICWs. We here describe a method to load cells with caged IP3 by local electroporation of monolayer cell cultures and to apply flash photolysis to increase intracellular IP3 and induce [Ca(2+)]i changes, or initiate ICWs. Moreover, the electroporation method allows loading of membrane-impermeable agents that interfere with IP3 and Ca(2+) signaling.
Department of Basic Medical Sciences, Physiology Group, Ghent University, 9000 Ghent, Belgium.
Elke
E
Decrock
Department of Basic Medical Sciences, Physiology Group, Ghent University, 9000 Ghent, Belgium.
Marijke
M
De Bock
Department of Basic Medical Sciences, Physiology Group, Ghent University, 9000 Ghent, Belgium.
Nan
N
Wang
Department of Basic Medical Sciences, Physiology Group, Ghent University, 9000 Ghent, Belgium.
Mélissa
M
Bol
Department of Basic Medical Sciences, Physiology Group, Ghent University, 9000 Ghent, Belgium.
Ashish K
AK
Gadicherla
Department of Basic Medical Sciences, Physiology Group, Ghent University, 9000 Ghent, Belgium.
Luc
L
Leybaert
D000255:Adenosine Triphosphate / Q000378:metabolism; D020013:Calcium Signaling; D018274:Electroporation / Q000379:methods*; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000528:radiation effects; D010782:Photolysis / Q000528:radiation effects*
C061868:inositol 1,4,5-trisphosphate 1-(2-nitrophenyl)ethyl ester; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate