Biology Reference
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a smaller companion (the calmodulin) in a pouch, can bounce high in signal and
may spawn improved progeny (
Baird
et al.
, 1999
) The increase in fluorescence
intensity after addition of histamine to Camgaroo-1 expressing HeLa cells was a
modest 40% and the characteristic calcium spiking activity was almost invisible, so
the sensor is not quite as bouncy as its name implies when sensing cytoplasmic free
calcium; however, addition of ionomycin caused an overall sevenfold increase in
fluorescence. The modest increase observed in response to histamine is almost
certainly due to the 7
m
M
K
0
d
, high relative to the calcium increase from around
100 nM to 1
m
M expected when histamine is added to HeLa cells.
Camgaroo-1 does not fold well at 37
C and could not be targeted to intracellu-
lar organelles, for example, mitochondria (
Baird
et al.
, 1999
). In an attempt to live
up to another of its attributes, the possibility that it may spawn improved progeny,
Camgaroo-1 was subjected to error-prone PCR mutagenesis (
Griesbeck
et al.
,
2001
); selection of the brightest clone after expression in
E. coli
revealed a point
mutation of residue 69 to methionine. This new sensor, Camgaroo-2, had
very similar calcium-binding properties and fluorescence dynamic range as
Camgaroo-1, but expressed far more brightly in HeLa cells grown at 37
C. The
response to histamine a (5% fluorescence increase) was lower even than for Cam-
garoo-1, but targeting to mitochondria using the targeting sequence of subunit
VIII of cytochrome
c
oxidase was demonstrated. Mitochondrial calcium increases
that raised the resting fluorescence signal by about 70% were demonstrated in
response to histamine and subsequent addition of ionopmycin gave an overall 1.5-
fold increase in fluorescence signal (
Griesbeck
et al.
, 2001
), lower than that ob-
served with cytoplasmic Camgaroo-2, perhaps because the resting mitochondrial
calcium concentration is higher than that of the cytoplasm.
Using a similar camgaroo-like strategy, the EF hand calcium-binding site was
introduced into EGFP between residues 144-145, 157-158, or 172-173 (
Zou
et al.
,
2007
). These Ca-G family sensors had extinction coe
Y
cients and quantum yields
comparable to EGFP. They operate in the ratiometric mode and with excitation at
398 and 490 nm showed a sensor dynamic range of 1.8 at a 510-nm emission
wavelength. Comprising a single EF hand-binding site, the apparent dissociation
constants are in the millimolar range (0.4-2 mM) and are, therefore, suitable only
for monitoring high calcium environments such as the ER. They are markedly pH
sensitive, with a p
K
a
of around 7.5. Expressed in the ER of HeLa and BHK-21cells,
they showed modest ratio changes in response to agonists (
Zou
et al.
, 2007
).
C. Pericam G-CaMP Family
1. Pericams
In pursuit of the idea that the clefts introduced into the beta can structure by
circular permutation might make the fluorophore more accessible to solution
protons and so susceptible to structural changes brought about by reorientation
of concatenated peptides, Miyawaki's group developed the pericam series of
