FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively.

The inhibitory effect of KN-93 and KN-62 as a result of CaM-directed blocking animal CaMK2 and plant CDPK activation

Aim. The goal of the study was determine whether, from a molecular point of view, inhibitors KN-93 and KN-62 are capable to disrupt the functioning of plant homologs of CaMK2 and being used as tools for the experimental study of Ca2+-dependent phosphorylation in higher plants. Methods. Selected calmodulin 1 H.sapiens reference structure and reconstructed spatial structure of calcium-binding domain of CPK1 A.thaliana. We have conducted the molecular docking of calmodulin-mediated inhibitors to CaMK2: KN-93 and KN-62 for CALM1 H. sapiens and calcium-binding domain of CPK1 A. thaliana with full ligand mobility and static amino acid residues with the use of CCDC GOLD Suite. Results. We have established the presence of spatially homologous structures within CALM1 and CPK1 that might be the binding sites for KN-93 and KN-62. Performing the molecular docking we have shown the utility of those pockets from the point of binding energy for KN-93 and KN-62. We have conducted comparative analysis basing on the results of the CCDC GOLD Suite score functions (GoldScore and ASPScore). Conclusions. It has been shown that the inhibitors of animal CaMK2 - KN93 and KN62 are capable of interacting with the site of the CaM-like domain of the plant protein kinase CPK1 homologous to the similar site of animal calmodulin, which may impair its functionality. Keywords: CaMK2, CDPK, CPK1, protein kinases, molecular docking, KN-93, KN-62.