Calcium/calmodulin-dependent protein kinase IIdelta 2 and gamma isoforms regulate potassium currents of rat brain capillary endothelial cells under hypoxic conditions.
Endothelial K+ and Ca2+ homeostasis plays an important role in the regulation of tissue supply and metabolism under normal and pathological conditions. However, the exact molecular mechanism of how Ca2+ is involved in the regulation of K+ homeostasis in capillary endothelial cells, especially under oxidative stress, is not clear. To reveal Ca2+-triggered pathways, which modulate K+ homeostasis, Ca2+/calmodulin-dependent protein kinase II and voltage-gated outward K+ currents were studied in rat brain capillary endothelial cells under hypoxia. Whole cell voltage-clamp measurements showed voltage-gated outward K+ current with transient and sustained components. mRNA and protein of Ca2+/calmodulin-dependent protein kinase II delta2 and two gamma isoenzymes were identified. Activation of the isoforms (autophosphorylation) was typically achieved by the Ca2+ ionophore ionomycin, which was prevented by the Ca2+/calmodulin-dependent protein kinase II-specific inhibitor KN-93. Hypoxia resulted in autophosphorylation of the delta2 and gammaB isoforms, augmented the current amplitude, increased the inactivation time constant, and decreased the extent of inactivation of the transient current. KN-93 prevented both the activation of the isoforms and the alterations in the K+ current characteristics. It is concluded that the activation of Ca2+/calmodulin-dependent protein kinase II decreases inactivation of the voltage-gated outward K+ current, thereby counteracting depolarization of the hypoxic endothelium.
Balla
Zsolt
Z
Forschungsinstitut für Molekulare Pharmakologie, Berlin, 13125 Germany.
Hoch
Brigitte
B
Karczewski
Peter
P
Blasig
Ingolf E
IE
D000818:Animals; D001921:Brain / Q000201:enzymology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry*; D002196:Capillaries / Q000201:enzymology*; D002460:Cell Line; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000201:enzymology*; D000860:Hypoxia; D015151:Immunoblotting; D008858:Microscopy, Phase-Contrast; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015221:Potassium Channels; D020033:Protein Isoforms; D012333:RNA, Messenger; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517677:Camk2d protein, rat; D011188:Potassium