Ischemia-Induced Neuronal Damage: A Role for Calcium/Calmodulin-Dependent Protein Kinase II

Calcium/calmodulin-dependent protein kinase II (CaM-kinase) is a central enzyme in regulating neuronal processes. Imbalances in the activity and distribution of this enzyme have been reported following in vivo ischemia, and sustained decreases in activity correlate with subsequent neuronal death. In this report, mice that had been rendered deficient in the alpha subunit of CaM-kinase using gene knock-out technology were utilized to determine whether this enzyme is causally related to ischemic damage. Using a focal model of cerebral ischemia, we showed that homozygous knock-out mice lacking the alpha subunit exhibited an infarct volume almost twice that of wild-type litter mates. Heterozygous mice exhibited slightly less damage following ischemia than did homozygous mice, but infarct volumes remained significantly larger than those of wild-type litter mates. We conclude that reduced amounts of the alpha subunit of CaM-kinase predisposes neurons to increased damage following ischemia and that any perturbation that decreases the amount or activity of the enzyme will produce enhanced susceptibility to neuronal damage.

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Alpha-subunit of calcium/calmodulin-dependent protein kinase II enhances gamma-aminobutyric acid and inhibitory synaptic responses of rat neurons in vitro

Journal of Neurophysiology ◽

10.1152/jn.1995.73.5.2099 ◽

1995 ◽

Vol 73 (5) ◽

pp. 2099-2106 ◽

Cited By ~ 49

Author(s):

R. A. Wang ◽

G. Cheng ◽

M. Kolaj ◽

M. Randic

Keyword(s):

Protein Kinase ◽

Gabaa Receptor ◽

Aminobutyric Acid ◽

Alpha Subunit ◽

Gamma Aminobutyric Acid ◽

Patch Clamp Technique ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Protein Kinase Ii ◽

Dependent Protein

1. Here we report that in acutely isolated rat spinal dorsal horn neurons, the gamma-aminobutyric acid-A (GABAA) receptor can be regulated by calcium/calmodulin-dependent protein kinase II (CaM-KII). Intracellularly applied, the alpha-subunit of CaM-KII enhanced GABAA-receptor-activated current recorded with the use of the whole cell patch-clamp technique. This effect was associated with reduced desensitization of GABA responses. 2. GABA-induced currents are also potentiated by calyculin A, an inhibitor of protein phosphatases 1 and 2A. 3. Conventional intracellular recordings were made from hippocampal CA1 neurons in slices to determine the effect of intracellular application of CaM-KII on inhibitory synaptic potentials evoked by electrical stimulation of the stratum oriens/alveus. The inhibitory synaptic potential was enhanced by CaM-KII; this mechanism may contribute to long-term enhancement of inhibitory synaptic transmission and may also play a role in other forms of plasticity in the mammalian brain.

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Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.92.5.1659 ◽

1995 ◽

Vol 92 (5) ◽

pp. 1659-1663 ◽

Cited By ~ 16

Author(s):

N. J. Olson ◽

T. Masse ◽

T. Suzuki ◽

J. Chen ◽

D. Alam ◽

...

Keyword(s):

Protein Kinase ◽

Alpha Subunit ◽

Functional Identification ◽

Dependent Protein Kinase ◽

Gene Encoding ◽

Calcium Calmodulin Dependent ◽

Protein Kinase Ii ◽

Dependent Protein

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Global Forebrain Ischemia Results in Decreased Immunoreactivity of Calcium/Calmodulin-Dependent Protein Kinase II

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1992.109 ◽

1992 ◽

Vol 12 (5) ◽

pp. 784-793 ◽

Cited By ~ 32

Author(s):

Severn B. Churn ◽

Amy Yaghmai ◽

John Povlishock ◽

Azhar Rafiq ◽

Robert J. DeLorenzo

Keyword(s):

Monoclonal Antibody ◽

Protein Kinase ◽

Neuronal Cell ◽

Cam Kinase Ii ◽

Forebrain Ischemia ◽

Cam Kinase ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Protein Kinase Ii ◽

Dependent Protein

Previous studies utilizing crude brain homogenates have shown that forebrain ischemia results in inhibition of calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity without large-scale proteolysis of the enzyme. In this report, a monoclonal antibody (1C3-3D6) directed against the β- (60-kDa) subunit of CaM kinase II that does not recognize ischemically altered enzyme was utilized to further investigate the ischemia-induced inhibition of CaM kinase II. Immunohistochemical investigations showed that the ischemia-induced decreased immunoreactivity of CaM kinase II occurred immediately following ischemic insult in ischemia-sensitive cells such as pyramidal cells of the hippocampus. No decrease in CaM kinase II immunoreactivity was observed in ischemia-resistant cells such as granule cells of the dentate gyrus. The decreased immunoreactivity was observed for CaM kinase II balanced for protein staining and calmodulin binding in vitro. In addition, autophosphorylation of CaM kinase II in the presence of low (7 μ M) or high (500 μ M) ATP did not alter immunoreactivity of the enzyme with 1C3-3D6. The data demonstrate the production of a monoclonal antibody that recognizes the β-subunit of CaM kinase II in a highly specific manner, but does not recognize ischemic enzyme. Together with previous studies, the data support the hypothesis that rapid, ischemia-induced inhibition of CaM kinase II activity may be involved in the cascade of events that lead to selective neuronal cell loss in stroke.

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A Purine Analog Kinase Inhibitor, Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor 59, Reveals a Role for Calcium/Calmodulin-Dependent Protein Kinase II in Insulin-Stimulated Glucose Transport

Endocrinology ◽

10.1210/en.2006-0446 ◽

2007 ◽

Vol 148 (1) ◽

pp. 374-385 ◽

Cited By ~ 11

Author(s):

Nicky Konstantopoulos ◽

Seb Marcuccio ◽

Stella Kyi ◽

Violet Stoichevska ◽

Laura A. Castelli ◽

...

Keyword(s):

Protein Kinase ◽

Glucose Uptake ◽

Glucose Transport ◽

Kinase Inhibitor ◽

Glut4 Translocation ◽

Wild Type ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Protein Kinase Ii ◽

Dependent Protein

Olomoucine is known as a cyclin-dependent kinase inhibitor. We found that olomoucine blocked insulin’s ability to stimulate glucose transport. It did so without affecting the activity of known insulin signaling proteins. To identify the olomoucine-sensitive kinase(s), we prepared analogs that could be immobilized to an affinity resin to isolate binding proteins. One of the generated analogs inhibited insulin-stimulated glucose uptake with increased sensitivity compared with olomoucine. The IC50 for inhibition of insulin-stimulated glucose uptake occurred at analog concentrations as low as 0.1 μm. To identify proteins binding to the analog, [35S]-labeled cell lysates prepared from 3T3-L1 adipocytes were incubated with analog chemically cross-linked to a resin support and binding proteins analyzed by SDS-PAGE. The major binding species was a doublet at 50–60 kDa, which was identified as calcium/calmodulin-dependent protein kinase II (CaMKII) by N-terminal peptide analysis and confirmed by matrix-assisted laser desorption ionization-mass spectrometry as the δ- and β-like isoforms. To investigate CaMKII involvement in insulin-stimulated glucose uptake, 3T3-L1 adipocytes were infected with retrovirus encoding green fluorescent protein (GFP)-hemagluttinin tag (HA)-tagged CaMKII wild-type or the ATP binding mutant, K42M. GFP-HA-CaMKII K42M cells had less kinase activity than cells expressing wild-type GFP-HA-CaMKII. Insulin-stimulated glucose transport was significantly decreased (∼80%) in GFP-HA-CaMKII K42M cells, compared with nontransfected cells, and cells expressing either GFP-HA-CaMKII or GFP-HA. There was not a concomitant decrease in insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII K42M cells when compared with GFP-HA alone. However, insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII cells was significantly higher, compared with either GFP-HA or GFP-HA-CaMKII K42M cells. Our results implicate the involvement of CaMKII in glucose transport in a permissive role.

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