Abstract
Genetically encoded calcium indicators (GECIs) are useful reporters of cell-signaling, neuronal, and network activities. We have generated novel fast variants and investigated the kinetic mechanisms of two recently developed red-fluorescent GECIs (RGECIs), mApple-based jRGECO1a and mRuby-based jRCaMP1a. In the formation of fluorescent jRGECO1a and jRCaMP1a complexes, calcium binding is followed by rate-limiting isomerization. However, fluorescence decay of calcium-bound jRGECO1a follows a different pathway from its formation: dissociation of calcium occurs first, followed by the peptide, similarly to GCaMP-s. In contrast, fluorescence decay of calcium-bound jRCaMP1a occurs by the reversal of the on-pathway: peptide dissociation is followed by calcium. The mechanistic differences explain the generally slower off-kinetics of jRCaMP1a-type indicators compared with GCaMP-s and jRGECO1a-type GECI: the fluorescence decay rate of f-RCaMP1 was 21 s−1, compared with 109 s−1 for f-RGECO1 and f-RGECO2 (37 °C). Thus, the CaM–peptide interface is an important determinant of the kinetic responses of GECIs; however, the topology of the structural link to the fluorescent protein demonstrably affects the internal dynamics of the CaM–peptide complex. In the dendrites of hippocampal CA3 neurons, f-RGECO1 indicates calcium elevation in response to a 100 action potential train in a linear fashion, making the probe particularly useful for monitoring large-amplitude, fast signals, e.g. those in dendrites, muscle cells, and immune cells.
Highlights
Encoded calcium indicators (GECIs) are useful reporters of cell-signaling, neuronal, and network activities
The absorption peak at 450 nm in the absence of Ca2ϩ is assigned to the neutral state of the chromophore for all red-fluorescent GECIs (RGECIs)
The advantage of the fast response kinetics of the probes is clearly seen in hippocampal CA3 pyramidal cells, in which rapid Ca2ϩ transients are used to visualize action potential firing. 10 Backpropagating action potentials (bAP) were detected by our fast variants, with an up to 4-fold faster decay rate (off of 77 ms for f-RGECO2) compared with jRGECO1a
Summary
Leon) [4] were followed by single-fluorophore sensors based on circularly permuted (cp) fluorescent proteins (FPs) (GCaMP, RCaMP, and Flash-pericam), which were better suited for twophoton imaging [5, 6]. Further mutation of the cpFP or replacement with other red fluorescent proteins led to the generation of variants with different excitation and emission spectra (B-CaMP, B-GECO, YCaMP, CyCaMP, etc.) [7, 8]. Those probes are potentially useful for multicolor imaging, as well as optogenetic experiments, because their emission wavelength does not overlap with the blue light used to excite light-gated channels or pumps that are simultaneously expressed (9 –11). Helassa et al [24] and Sun et al [21] proposed reaction mechanisms for the formation of the fluorescent state of GCaMP-s In both models Ca2ϩ binding to the N-lobe of. The novel variant RGECI probes were analyzed in detail and showed significantly faster transients than their parental variants in ATP-
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