Biology Reference
In-Depth Information
Abstract
The quantitative manipulation of intracellular calcium concentration ([Ca
2
þ
]
i
)isa
valuable instrument in the modern cell biologists' toolbox for unraveling the many
cell processes controlled by calcium. I summarize here the major classes of photo-
sensitive calcium chelators used to elevate or reduce [Ca
2
þ
]
i
,withanemphasison
their physicochemical properties and methods of calculating magnitudes and kinet-
ics of e
ects on [Ca
2
þ
]
i
of flashes and steady light, in order to encourage the choice of
the best substance for particular applications. The selection and calibration of
appropriate light sources, and procedures for introducing the chelators into cells,
spatially restricting [Ca
2
þ
]
i
changes, and measuring the profiles of [Ca
2
þ
]
i
changes
imposed by photolysis, are also described. The final section describes a selection of
biological applications.
V
I. Introduction
Photolabile Ca
2
þ
chelators, sometimes called caged Ca
2
þ
chelators, are used to
control [Ca
2
þ
]
i
in cells rapidly and quantitatively. A beam of light is aimed at cells
filled with a photosensitive substance that changes its a
nity for binding Ca
2
þ
.
Several such compounds have been invented that allow the e
Y
V
ective manipulation
of [Ca
2
þ
]
i
in cells. These compounds o
er tremendous advantages over the alter-
native methods of microinjecting Ca
2
þ
salts, pharmacologically releasing Ca
2
þ
from intracellular stores, or increasing cell membrane permeability to Ca
2
þ
using
ionophores, detergents, electroporation, fusion with micelles, or activation of
voltage-dependent channels, in terms of specificity of action, repeatability and
reliability of e
V
V
ect, maintenance of cellular integrity, definition of spatial extent,
and rapidity of e
ect, all combined with the ability to maintain the [Ca
2
þ
]
i
change
V
for su
cient time to measure its biochemical or physiological consequences. Only
photosensitive chelators allow the concentration of Ca
2
þ
in the cytoplasm of intact
cells to be changed rapidly by a predefined amount over a selected region or over
the whole cell. Since loading can precede photolysis by a substantial amount of
time, cells can recover from the adverse e
Y
ects of the loading procedure before the
experiments begin. The ideal photosensitive Ca
2
þ
chelator does not exist, but
would have the following properties.
V
1. The compound could be introduced easily into cell, by microinjection or by
loading a membrane-permeating derivative that would be altered enzymatically to
an impermeant version trapped in cells.
2. The compound could be loaded withCa
2
þ
to such a level that the unphotolyzed
formwould bu
er the [Ca
2
þ
]
i
to near the normal resting level, so its introduction into
cells would not perturb the resting Ca
2
þ
level. Additionally, by adjusting the Ca
2
þ
loading or selecting chelator variants, the initial resting Ca
2
þ
level could be set to
somewhat higher or lower than the normal resting concentration.
V
