Biomedical Engineering Reference
In-Depth Information
and Pieribone
(93)
have developed a similar construct, SPARC,
with very rapid kinetics. Unfortunately, in mammalian cells,
both of these FP-voltage sensors are only minimally expressed in
the extracellular membrane
(94)
. They appear to be retained in
the endoplasmic reticulum. Thomas Knopfel and collaborators
(95)
investigated a new class of voltage- sensitive proteins,
the voltage-sensitive phosphatase from
Ciona intestinalis (96)
.
They replaced the phosphatase domain with a FRET pair and
this protein, VSFP2.1, both trafficked well to the external
membrane of hippocampal neurons and had a voltage-dependent
response. VFSP2.1 was faster than FlaSh but slower than SPARC.
Future efforts will be needed to improve the response time and
signal size of VFSP2.1 and to test the construct in transgenic
systems.
There exists a rather large family of genetically encoded fluores-
cent Ca
2
+
indicator proteins. Until recently, all protein calcium
sensors employed Ca
2
+
binding to calmodulin (CaM) and Ca
2
+
-
dependent interaction of calmodulin and the CaM binding pep-
tide M13 as a calcium sensor. The CaM-M13 complex was then
attached to green fluorescent protein or one of its variants to gen-
erate a single-wavelength Ca
2
+
indicator
(97-102)
. Alternatively,
the CaM-M13 complex was sandwiched between CFP (cyan flu-
orescent protein) and YFP (yellow fluorescent protein) to gen-
erate ratiometric Ca
2
+
sensors like the “Cameleons”
(103, 104)
.
Calmodulin-based calcium sensors function well in invertebrates
and lower vertebrates (for review see refs. in Miyawaki
(104)
), but
show rather poor performance in mammals
(105-108)
. Significant
improvement of protein calcium sensor performance in mam-
malian neurons came from the use of alternative Ca
2
+
-binding
protein Troponin C (TnC;
(109-111)
). The members of the TnC
family have relatively high dynamic range (4-fold increase in sig-
nal strength upon changing Ca
2
+
concentration from 0 to 10
mM), rather fast rise and decay times
(108)
and they respond
linearly to an increase in Ca
2
+
concentration within the physi-
ological activity range
(102, 111)
. TnC-based indicators can be
expressed transgenically in mice and allow in vivo imaging of neu-
ral function with single-cell and even subcellular resolution
(111)
.
While recent developments significantly improved the properties
of protein calcium sensors, when compared to organic Ca
2
+
indi-
cators, the protein sensors are still inferior when Ca
2
+
sensitivity
and/or dynamic range of the indicator are considered (see Table
1 in Garaschuk et al.
(108)
).
Taken together, optical recordings already provide unique
insights into brain activity and organization. Improvements in
sensitivity or selectivity would make these methods even more
powerful.
5.3.4. Calcium Sensors
