Activation of Ca2+/calmodulin-dependent protein kinase II by extracellular calcium in cultured hippocampal pyramidal neurons.
The ability of various stimuli to convert Ca2+/calmodulin-dependent protein kinase II (CaMKII) into a Ca2+-independent (autonomous) form was examined in cultured embryonic rat hippocampal pyramidal neurons. The most effective stimulation by far was observed when cells were equilibrated in buffer containing low extracellular [Ca2+] ([Ca2+]o) (approximately 50 nM) and then shifted to normal [Ca2+]o (approximately 1.26 mM) by addition of CaCl2 (referred to as "Ca2+ stimulation"). Virtually complete (>90%) conversion of the kinase to the autonomous form occurred within 30-50 s, with a return to baseline within 5 min. By contrast, depolarization of cells with high [K+] or treatment with glutamate or a Ca2+ ionophore caused insignificant increases (<10%) in levels of the autonomous form. The Ca2+-stimulated increase in CaMKII autonomy coincided with a two- to threefold increase in kinase subunit phosphorylation. In the first 40 s of Ca2+ stimulation, 32P incorporation into the immunoprecipitated subunits of CaMKII occurred exclusively on threonine residues, including Thr286Thr287 of the alpha/beta subunits. Longer incubation of cells resulted in a decline of phosphothreonine content, whereas levels of phosphoserine-containing peptides showed a significant increase. The activation of CaMKII by Ca2+ stimulation was accompanied by only a small rise in intracellular [Ca2+]. Inhibitor studies showed that Na+-dependent action potentials and Ca2+ influx through glutamate receptors or voltage-sensitive Ca2+ channels did not contribute to the activation. Moreover, CaMKII was not activated by extracellular addition of other cations, including Mn2+, Mg2+, Co2+, or Gd3+. Although the mechanism of Ca2+ stimulation is presently unclear, it may involve either activation of extracellular calcium receptors or capacitative calcium entry. The dramatic rise in CaMKII autonomy and the Ca2+ selectivity of the response suggest a direct and specific relationship between [Ca2+]o and the state of activation of the kinase in intact neurons.
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637, USA.
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Scholz
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Palfrey
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D017966:Pyramidal Cells / Q000201:enzymology*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology
D002413:Cations, Divalent; D010748:Phosphopeptides; D018698:Glutamic Acid; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium