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were used to identify brighter GCaMP2 mutants; attention was also paid to
improving the sensitivity to small calcium changes through mutations of the
CaM EF hands and of the M13/CaM interaction domains. The upshot was
GCaMP3, with a dynamic range of 12, due to a twofold decrease in calcium-free
fluorescence and a 1.5-fold increase in calcium-saturated fluorescence relative to
GCaMP2, and a
K
0
d
of 0.66
m
M(
Tian
et al.
, 2009
).
3. Cases 12 and 16
The Case (presumably Calcium
se
nsor) constructs were developed by analyzing
the linker sequences between M13 and cpEYFP/GFP and cpEYFP/GFP and
calmodulin and the three key residues 148, 145, and 203 in the pericams and
G-CaMPs (
Souslova
et al.
, 2007
). Based on this analysis, constructs were made
containing the G-CaMP linker sequences and the cpEYFP derived from Ratio-
metric Pericam. Nine point mutants were made with alterations in both the linker
sequences and in the three key residues within cpEYFP. As expected, combinations
of Asp148 and Phe203 produced ratiometric indicators akin to Ratiometric Peri-
cam, while Asn or Glu at residue 148 combined with Phe203 had a single excitation
peak at 490 nm. The Glu148/Thr145 and Glu148/S145 variants showed a 14.5-fold
increase in 490-nm fluorescence between calcium-free and calcium-bound forms.
The E148/S145 variant of these pericam-G-CaMP hybrids was optimized for
folding at 37
C using error-prone PCR, resulting in a variant with a 12-fold
dynamic range named Case12. Substituting Thr for Ser at the 145 position of
Case12 gave Case16, with a 16.5-fold dynamic range. The apparent dissociation
constant for both Cases 12 and 16 was 1
m
M. Like the pericams and G-CaMP
sensors, the calcium-bound forms of Cases 12 and 16 (p
K
a
7.2)—and thus their
fluorescence—are a
V
ected by any changes in pH within the physiological range.
III. Applications of Genetically Encoded Sensors
A. Targeting to Subcellular Locations
Low molecular mass fluorescent calcium sensors do make their way to intracel-
lular compartments (
Silver
et al.
, 1992
) and can be used to measure calcium there,
but they are di
cult to target precisely (
Varadi and Rutter, 2002b
). One of the two
major advantages of genetically encoded calcium sensors is that chimeric con-
structs and signaling tags can target them specifically to subcellular locations.
Methods to achieve some of these specific localizations had already been developed
for GFP itself and for the calcium sensor aequorin (
De Giorgi
et al.
, 1996
). The
ability to target cameleons YC-3er and YC-4er was demonstrated in the study in
which cameleons were first described (
Miyawaki
et al.
, 1997
).
Y
