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• W2910175656 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. 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." @default.
• W2910175656 created "2019-01-25" @default.
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• W2910175656 date "2019-03-01" @default.
• W2910175656 modified "2023-10-16" @default.
• W2910175656 title "The kinetic mechanisms of fast-decay red-fluorescent genetically encoded calcium indicators" @default.
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• W2910175656 doi "https://doi.org/10.1074/jbc.ra118.004543" @default.
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