Visualization of synaptic Ca2+ /calmodulin-dependent protein kinase II activity in living neurons.
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.
Takao
Keizo
K
RIKEN-Massachusetts Institute of Technology (MIT) Neuroscience Research Center, The Picower Center for Learning and Memory, Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts 02139, USA.
Okamoto
Ken-Ichi
K
Nakagawa
Terunaga
T
Neve
Rachael L
RL
Nagai
Takeharu
T
Miyawaki
Atsushi
A
Hashikawa
Tsutomu
T
Kobayashi
Shigeo
S
Hayashi
Yasunori
Y
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002460:Cell Line; D003952:Diagnostic Imaging / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015870:Gene Expression / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007313:Insecta; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008967:Molecular Biology / Q000379:methods; D016297:Mutagenesis, Site-Directed / Q000379:methods; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011810:Quinoxalines / Q000494:pharmacology; D026941:Sodium Channel Blockers / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D013569:Synapses / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection / Q000379:methods; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology; D000096926:Benzenesulfonamides
D015763:2-Amino-5-phosphonovalerate; D000255:Adenosine Triphosphate; D001596:Benzylamines; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D018698:Glutamic Acid; D005998:Glycine; D008164:Luminescent Proteins; D016202:N-Methylaspartate; D011810:Quinoxalines; D026941:Sodium Channel Blockers; D013449:Sulfonamides; D013779:Tetrodotoxin; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; C425780:KN 92; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; C072105:KN 93; D000096926:Benzenesulfonamides