Topic Introduction
Electroporation Loading and Flash Photolysis to
Investigate Intra- and Intercellular Ca
2+
Signaling
Elke Decrock,
1
Marijke De Bock,
1
Nan Wang, Mélissa Bol, Ashish K. Gadicherla,
and Luc Leybaert
2
Department of Basic Medical Sciences, Physiology Group, Ghent University, 9000 Ghent, Belgium
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 neigh-
boring cells. The intracellular messenger inositol 1,4,5-trisphosphate (IP
3
) 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” IP
3
into cells followed by photolytic “uncaging” with UV light,
thereby liberating IP
3
, 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 IP
3
by local electro-
poration of monolayer cell cultures and to apply flash photolysis to increase intracellular IP
3
and
induce [Ca
2+
]
i
changes, or initiate ICWs. Moreover, the electroporation method allows loading of
membrane-impermeable agents that interfere with IP
3
and Ca
2+
signaling.
FROM INTRA- TO INTERCELLULAR Ca
2+
SIGNALING
The importance of Ca
2+
as a second messenger in the cell is emphasized by the observation that even
the most primitive prokaryotes express a variety of active Ca
2+
pumps and passive channels or
transporters that, respectively, create and dissipate the Ca
2+
gradient across the plasma membrane.
The multiplicity of messengers and channels existing today brings up a picture of a highly complex
Ca
2+
signaling toolkit tuned to fulfill individual cellular needs throughout vertebrate life, from fertil-
ization through development to death and including the processes of cell proliferation, differentiation,
neurotransmitter release, secretion, synaptic plasticity, gene expression, immune responses, muscle
contraction, cardiomyocyte function, endothelial permeability, apoptosis, and many others (Berridge
1997; Case et al. 2007; Iino 2010).
All eukaryotic cells control their intracellular, cytosolic Ca
2+
concentration ([Ca
2+
]
i
) through an
intimate interplay between (i) Ca
2+
entry from the extracellular space, (ii) Ca
2+
release from intra-
cellular storage sites, (iii) Ca
2+
sequestration into the endoplasmic reticulum (ER) and other organ-
elles, and (iv) Ca
2+
extrusion out of the cells. The free [Ca
2+
]
i
in resting cells varies between 50 nM and
1
These two authors contributed equally.
2
Correspondence: luc.leybaert@ugent.be
© 2015 Cold Spring Harbor Laboratory Press
Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top066068
239
Cold Spring Harbor Laboratory Press
on April 7, 2016 - Published by http://cshprotocols.cshlp.org/ Downloaded from