Inhibition of phosphatase activity facilitates the formation and maintenance of NMDA-induced calcium/calmodulin-dependent protein kinase ii clusters in hippocampal neurons

• Published in: Neuroscience 2005, Vol.130 (3), p.651-656
• Main Authors: Tao-Cheng, J.-H., Vinade, L., Winters, C.A., Reese, T.S., Dosemeci, A.
• Format: Article
• Language: English
• Subjects: Animals; autophosphorylation; Biological and medical sciences; Calcium-Calmodulin-Dependent Protein Kinases - biosynthesis; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; calcium/calmodulin-dependent protein kinase II; calyculin A; Cytoplasm - enzymology; Enzyme Inhibitors - pharmacology; Excitatory Amino Acid Agonists - pharmacology; Fundamental and applied biological sciences. Psychology; Hippocampus - cytology; Hippocampus - drug effects; Hippocampus - enzymology; immunogold electron microscopy; Immunohistochemistry; Isoenzymes - biosynthesis; Isoenzymes - metabolism; N-Methylaspartate - pharmacology; Neurons - drug effects; Neurons - enzymology; okadaic acid; Paraffin Embedding; Phosphoric Monoester Hydrolases - antagonists & inhibitors; Phosphorylation; Rats; Vertebrates: nervous system and sense organs
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2004.10.008

The majority of hippocampal neurons in dissociated cultures and in intact brain exhibit clustering of calcium/calmodulin-dependent protein kinase II (CaMKII) into spherical structures with an average diameter of 110 nm when subjected to conditions that mimic ischemia and excitotoxicity [Neuroscience 106 (2001) 69]. Because clustering of CaMKII would reduce its effective concentration within the neuron, it may represent a cellular strategy to prevent excessive CaMKII-mediated phosphorylation during episodes of Ca 2+ overload. Here we employ a relatively mild excitatory stimulus to promote sub-maximal clustering for the purpose of studying the conditions for the formation and disappearance of CaMKII clusters. Treatment with 30 μM N-methyl- d-aspartic acid (NMDA) for 2 min produced CaMKII clustering in approximately 15% of dissociated hippocampal neurons in culture, as observed by pre-embedding immunogold electron microscopy. These CaMKII clusters could be labeled with antibodies specific to the phospho form (Thr286) of CaMKII, suggesting that at least some of the CaMKII molecules in clusters are autophosphorylated. To test whether phosphorylation is involved in the formation and maintenance of CaMKII clusters, the phosphatase inhibitors calyculin A (5 nM) or okadaic acid (1 μM) were included in the incubation medium. With inhibitors more neurons exhibited CaMKII clusters in response to 2 min NMDA treatment. Furthermore, 5 min after the removal of NMDA and Ca 2+, CaMKII clusters remained and could still be labeled with the phospho-specific antibody. In contrast, in the absence of phosphatase inhibitors, no clusters were detected 5 min after the removal of NMDA and Ca 2+ from the medium. These results suggest that phosphatases type 1 and/or 2A regulate the formation and disappearance of CaMKII clusters.