Sustained elevation of calcium induces Ca2+/calmodulin-dependent protein kinase II clusters in hippocampal neurons

Treatment of cultured hippocampal. neurons with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the absence of glucose mimics ischemic energy depletion and induces formation of Ca2+/calmodulin-d ependent protein kinase II (CaMKII) clusters, spherical structures with dia meters of 75-175 nm [Dosemeci et al., J. Neurosci. 20 (2000) 3076-3084]. Th e demonstration that CaMKII clustering occurs in the intact, adult rat brai n upon interruption of blood flow indicates that clustering is not confined to cell cultures. Application of N-methyl-Daspartate (250 muM, 15 min) to hippocampal cultures also induces cluster formation, suggesting a role for Ca2+. Indeed, intracellular Ca2+ monitored with Fluo3-AM by confocal micros copy reaches a sustained high level within 5 min of CCCP treatment. The app earance of immunolabeled CaMKII clusters, detected by electron microscopy, follows the onset of the sustained increase in intracellular Ca2+. Moreover , CaMKII does not cluster when the rise in intracellular Ca2+ is prevented by the omission of extracellular Ca2+ during CCCP treatment, confirming tha t clustering is Ca2+-dependent. A lag period of 1-2 min between the onset o f high intracellular Ca2+ levels and the formation of CaMKII clusters sugge sts that a sustained increase in Ca2+ level is necessary for the clustering . CaMKII clusters disappear within 2 h of returning the cultures to normal incubation conditions, at which time no significant cell death is detected. These results indicate that pathological conditions that promote sustained episodes of Ca2+ overload result in a transitory clustering of CaMKII into spherical structures. CaMKII clustering may represent a cellular defense me chanism to sequester a portion of the CaMKII pool, thereby preventing exces sive protein phosphorylation. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.