kn-93 and 2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the CNS and critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, because of a lack of specific methods. We tried to address this by optically detecting the conformational change in CaMKII during activation using fluorescence resonance energy transfer (FRET). The engineered FRET probe Camuialpha detects calmodulin binding and autophosphorylation at threonine 286 that renders the enzyme constitutively active. In combination with two-photon microscopy, we demonstrate that Camuialpha can be used to observe temporal and spatial regulation of CaMKII activity in living neurons.

Topics: 2-Amino-5-phosphonovalerate; Adenosine Triphosphate; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Analysis of Variance; Animals; Benzylamines; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Line; Diagnostic Imaging; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fluorescence Resonance Energy Transfer; Gene Expression; Glutamic Acid; Glycine; Hippocampus; Humans; Insecta; Luminescent Proteins; Microscopy, Confocal; Molecular Biology; Mutagenesis, Site-Directed; N-Methylaspartate; Neurons; Phosphorylation; Protein Binding; Quinoxalines; Sodium Channel Blockers; Sulfonamides; Synapses; Tetrodotoxin; Time Factors; Transfection