Protein kinase C isozyme-specific potentiation of expressed Ca v 2.3 currents by acetyl-beta-methylcholine and phorbol-12-myristate, 13-acetate.
Protein kinase C (PKC) is implicated in the potentiation of Ca v 2.3 currents by acetyl-beta-methylcholine (MCh), a muscarinic M1 receptor agonist or phorbol-12-myristate, 13-acetate (PMA). The PKC isozymes responsible for the action of MCh and PMA were investigated using translocation as a measure of activation and with isozyme-selective antagonists and siRNA. Ca v channels were expressed with alpha1 2.3, beta1b and alpha2delta subunits and muscarinic M1 receptors in the Xenopus oocytes and the expressed currents (I Ba) were studied using Ba2+ as the charge carrier. Translocation of PKC isozymes to the membrane studied by Western blot revealed that all eleven known PKC isozymes are present in the Xenopus oocytes. Exposure of the oocytes to MCh led to the translocation of PKC alpha whereas PMA activated PKC betaII and epsilon isozymes. The action of MCh was inhibited by Go 6976, an inhibitor of cPKC isozymes or PKC alpha siRNA. PMA-induced potentiation of Ca v 2.3 currents was inhibited by CG533 53, a PKC betaII antagonist, betaIIV5.3, a peptide translocation inhibitor of PKC betaII or PKC betaII siRNA. Similarly, epsilonV1.2, a peptide translocation inhibitor of PKC epsilon or PKC epsilon siRNA inhibited PMA action. The inhibitors of PKC increased the basal I Ba slightly. It is possible that some PKC isozymes have negative control over the I Ba. Our results implicate PKC alpha in the potentiation of Ca v 2.3 currents by MCh and PKC betaII and epsilon in the potentiation of Ca v 2.3 currents by PMA.
Rajagopal
Senthilkumar
S
Department of Anesthesiology, P.O. Box 800710, University of Virginia Health Science Systems, 1766 Lane Road, Charlottesville, VA 22908-0710, USA.
Fang
Hongyu
H
Patanavanich
Saharat
S
Sando
Julianne J
JJ
Kamatchi
Ganesan L
GL
D000818:Animals; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism; D020908:Calcium Channels, R-Type / Q000187:drug effects / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000187:drug effects / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D007527:Isoenzymes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D016210:Methacholine Chloride / Q000494:pharmacology*; D018721:Muscarinic Agonists / Q000494:pharmacology; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D043643:Receptor, Muscarinic M1 / Q000819:agonists* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014982:Xenopus laevis
C498916:Cacna1e protein, mouse; D015220:Calcium Channels; D020908:Calcium Channels, R-Type; D002273:Carcinogens; D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D018721:Muscarinic Agonists; D034741:RNA, Small Interfering; D043643:Receptor, Muscarinic M1; D016210:Methacholine Chloride; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate