Genetically Encoded Calcium Indicators Based on Troponin C and Fluorescent Proteins

Genetically encoded calcium probes allow the visualization and
quantification of intracellular calcium dynamics with great
specificity and sensitivity. Until now, all genetically encoded
calcium indicators have shared a common design that consists of
mutants of the green fluorescent protein (GFP) as fluorophores and
calmodulin as the calcium binding moiety, in several
configurations. However, most of these calmodulin-based probe types
show deficiencies such as reduced dynamic ranges when expressed
within transgenic organisms and a lack of calcium sensitivity in
certain subcellular targetings. A likely reason for this reduced
sensitivity is that calmodulin is an ubiquitous signal protein in
cell metabolism and thus stringently regulated. Thus, we chose to
develop novel types of calcium probes based on the muscle calcium
sensor troponin C, a protein that is not a constituent of
non-muscle cells and therefore less likely to interact with
cytosolic activities. By going through a series of cloning
optimization steps, a set of new ratiometric calcium indicators was
created using domains of skeletal and cardiac muscle troponin C
variants as calcium binding moieties. These constructs showed in
vitro FRET ratio changes of up to 140 %, had calcium dissociation
constants ranging from 470 nM to 29 µM, and were functional in
intracellular targetings in which previous indicators had failed.
The new indicators expressed homogenously with no signs of
aggregation in HEK293 cells as well as in rat hippocampal neurons,
and large and dynamic ratio changes could be quantified after drug
stimulation in cell culture. Membrane labeling experiments with the
indicator construct TN-L15 were successful in HEK293 cells and
hippocampal neurons. When targeted to the plasma membrane, the
indicator readily responded to agonist-induced increases in
cytosolic calcium and kept its full dynamic range. In the last part
of this work, transgenic mouse lines were created expressing one of
the new calcium indicators in the cytosol of neurons. Imaging
experiments in live tissue cultures and brain slices revealed
responses to rises in calcium that were superior to previously
published indicator performance in mouse lines expressing other
calcium probes. The novel troponin C-based probes of intracellular
calcium developed in this work have the potential for monitoring
calcium dynamics in applications in which previous
calmodulin-containing calcium indicators failed, possibly because
they interact less with the cellular biochemical machinery and are
thus more compatible with transgenic expression in tissue and whole
organisms.