Biology Reference
In-Depth Information
preceding the pericams' by a matter of months (
Nakai
et al.
, 2001
). Both the CaM-
cpGFP-M13 and M13-GFP-CaM concatenations were tested: only the latter
showed significant calcium-sensing properties. Twenty-six variants of the M13-
N149cpGFPC144-CaM concatenate were tested and the variant that showed the
greatest fluorescence increase in HEK-239 cells after ATP addition was termed
G-CaMP (presumably for green fluorescent calmodulin protein). In HEK-239
cells, G-CaMP gave a 1.5-fold increase in fluorescence in response to ATP and a
fourfold increase in response to ionomycin. G-CaMP has very similar fluorescence
parameters to Flash Pericam, with an excitation maximum at 489 nm, an emission
maximum at 509 nm and a 4.5-fold increase in fluorescence on calcium binding
(cf. eightfold for Flash Pericam). The apparent dissociation curve was monotonic,
with a
K
0
d
of 0.24
m
M. As with the camgaroos and pericams and for the same
reasons, the sensor signal is strongly pH dependent in the physiological range. The
association time constant for calcium binding was strongly calcium dependent and
varied from 250 ms at low calcium concentration to 2.5 ms at higher concentra-
tions; the dissociation time constant was 200 ms. G-CaMP expresses poorly at
37
C. G-CaMP-expressing smooth muscle showed a response to rapid depolari-
zation of around 50%, with a time course comparable to that previously measured
with Fluo-3. Carbachol addition gave a 2.5-fold increase. pH was monitored in
these experiments and did not change (
Nakai
et al.
, 2001
).
This first GCaMP family member, later designated GCaMP1, had very weak
fluorescence when expressed at physiological temperatures compared to GFP
itself. This was addressed by introducing two mutations V163A and S175G that
were known to improve the temperature-dependent maturation of GFP to give a
variant known as G-CaMP1.6 (
Ohkura
et al.
, 2005
); this increased brightness
about 40-fold. However, these modifications did not lead to adequate maturation
above 30
C. The G-CaMP construct was subjected to error-prone PCR mutagen-
esis and the clones fluorescing most brightly at 37
C were selected (
Tallini
et al.
,
2006
). The two new mutations in the brightest clone were identified (D180Y and
V93I), but it also turned out that the RSET leader sequence that had been added to
facilitate purification of the expressed protein was essential for thermal stability at
37
C. This construct, GCaMP2, is around 200 times brighter than G-CaMP1 at
37
C (with an extinction coe
cient at 487 nm of 19,000 and a quantum yield of
0.93 with emission at 508 nm) and shows the same four- to fivefold increase in
fluorescence a saturating calcium concentrations when compared to calcium-free
conditions. Though not reported, it should be assumed that this sensor remains
pH-sensitive. GCaMP2 was expressed using tissue-specific promoters in transgenic
animals and calcium transients were detected in granule cells in cerebellar slices
(
Diez-Garcia
et al.
, 2005
) and in isolated heart
in vitro
and in adult and embryonic
heart
in vivo
(
Tallini
et al.
, 2006
). Some insight into the sensor mechanism of
GCaMP2 is a
Y
V
orded by its crystal structure (
Akerboom
et al.
, 2009; Wang
et al.
, 2008
).
Even so, in HEK293 cells, GCaMP2 fluorescence is still 100-fold lower than
GFP itself (
Tian
et al.
, 2009
). HEK293 cell medium-throughput screening assays
