S2-5 Molecular mechanisms of neuronal degeneration in neurodegenerative diseases

S6 MOLECULAR

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To elucidate the molecular mechanisms of neurodegeneration questions come out; 1. What are the molecular mechanisms of

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Symposium 3. Progress in imaging second messengers

s3-1

VISUALIZATION OF CALCIUMCALMODULIN-DEPENDENT PROTEIN KINASE II ACTIVITY IN CULTURED HIPPOCAMPAL NEURONS. YOHIHISA KUDO AND HIDEYOSHI HIGASHI. Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-03 and Mitsubishi Kasei Institute of Life Sciences,Minamiooya, Machida, Tokyo 194 Japan. Our aimwas to visualizethe dynamicfeaturesof Ca2t/calmodulin-dependent protein kinaseII (CaMKII) activity in living cells. To do so, we have synthesizeda new reagent, AS2, by conjugatinga fluoroprobe, 6-acryloyl- 2-dimethylaminonaphthalene(acrylodan), an ideal probe for detectingconformationalchangesin proteins, to syntide 2, a specific peptide substratefor CaMKII. In cell-freeconditions,the conjugatewas found to be an effective indicatorof calmodulinactivationby Ca2+ and the subsequentactivation of CaMKII. KN-62, a CaMKII specific inhibitor, depressedthe fluorescencechange, while H-7, a proteinkinaseC andprotein kinaseA inhibitor had no effect on it. The reagentis cell-permeable and can stain living cellswhen bath-applied. Using this techniquewe were ableto obtainfluorescenceimagesof stainedcellsand analyze the dynamic featuresof CaMKII insidethe cellsby meanof imageprocessing. Regionalheterogeneityof CaMKII activation in culturedhippocampalneuronsfollowing Lglutamate administrationwas seen.‘Ihe effect was also blockedby KN-62. Although the indicatorseemedto be still incompletein presentform, we improved the indicatorto makeit muchsensitiveand much stableby changingthe amino acid sequences and the fluorescentprobes. Their availability as indicatorsfor CaMKII activity will be discussed.