title: Sustained elevation of calcium induces Ca(2+)/calmodulin-dependent protein kinase II clusters in hippocampal neurons.
abstract: 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 Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) clusters, spherical structures with diameters of 75-175 nm [Dosemeci et al., J. Neurosci. 20 (2000) 3076-3084]. The demonstration that CaMKII clustering occurs in the intact, adult rat brain upon interruption of blood flow indicates that clustering is not confined to cell cultures. Application of N-methyl-D-aspartate (250 microM, 15 min) to hippocampal cultures also induces cluster formation, suggesting a role for Ca(2+). Indeed, intracellular Ca(2+) monitored with Fluo3-AM by confocal microscopy reaches a sustained high level within 5 min of CCCP treatment. The appearance of immunolabeled CaMKII clusters, detected by electron microscopy, follows the onset of the sustained increase in intracellular Ca(2+). Moreover, CaMKII does not cluster when the rise in intracellular Ca(2+) is prevented by the omission of extracellular Ca(2+) during CCCP treatment, confirming that clustering is Ca(2+)-dependent. A lag period of 1-2 min between the onset of high intracellular Ca(2+) levels and the formation of CaMKII clusters suggests that a sustained increase in Ca(2+) 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 Ca(2+) overload result in a transitory clustering of CaMKII into spherical structures. CaMKII clustering may represent a cellular defense mechanism to sequester a portion of the CaMKII pool, thereby preventing excessive protein phosphorylation.
authors.affiliation: Laboratory of Neurobiology, NINDS, NIH, Bethesda, MD 20892, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Tao-Cheng
authors.forename: L
authors.initials: L
authors.lastname: Vinade
authors.forename: C
authors.initials: C
authors.lastname: Smith
authors.forename: C A
authors.initials: CA
authors.lastname: Winters
authors.forename: R
authors.initials: R
authors.lastname: Ward
authors.forename: M W
authors.initials: MW
authors.lastname: Brightman
authors.forename: T S
authors.initials: TS
authors.lastname: Reese
authors.forename: A
authors.initials: A
authors.lastname: Dosemeci
mesh_terms: D000367:Age Factors; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D018929:Cell Culture Techniques; D002478:Cells, Cultured / Q000201:enzymology / Q000473:pathology / Q000648:ultrastructure; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology / Q000473:pathology / Q000648:ultrastructure; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology*; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology; D005333:Fetus; D006624:Hippocampus / Q000201:enzymology* / Q000473:pathology / Q000503:physiopathology; D007150:Immunohistochemistry; D007424:Intracellular Fluid / Q000187:drug effects / Q000201:enzymology*; D008854:Microscopy, Electron; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000201:enzymology* / Q000473:pathology / Q000648:ultrastructure; D009498:Neurotoxins / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014475:Uncoupling Agents / Q000494:pharmacology
chemical_list: D002614:Chelating Agents; D018690:Excitatory Amino Acid Agonists; D009498:Neurotoxins; D014475:Uncoupling Agents; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D016202:N-Methylaspartate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium