Selective disruption by protein kinases of G-protein-mediated Ca2+ channel modulation.
1. We studied the effects of phorbol-12-myristate, 13-acetate (PMA) on G-protein-mediated inhibition of Ca2+ channels by several neurotransmitters in rat superior cervical ganglion (SCG) sympathetic neurons, with the use of the whole cell patch clamp. PMA attenuated membrane-delimited inhibition of calcium currents (ICa) by norepinephrine (NE) and somatostatin by more than half, but did not attenuate inhibition by M1 muscarinic receptors, which use a diffusible cytoplasmic messenger. Inhibition of ICa by NE through pertussis-toxin-sensitive and -insensitive G proteins was equally attenuated by PMA. PMA enhanced ICa in about half the neurons (enhancement of 10 +/- 1%, mean +/- SE) and strongly reduced the holding current in 44 of 61 cells. 2. The M-type K+ current (IM) was not suppressed by PMA, and PMA did not attenuate inhibition of IM by muscarinic agonists, which is also via a diffusible cytoplasmic messenger. 3. Attenuation of NE and somatostatin inhibition by PMA was blocked by 1 microM staurosporine, a broad-spectrum protein kinase inhibitor. Tests with three inhibitors selective for distinct isoforms of protein kinase C (PKC) gave mixed results. PMA's actions were unaffected by 1 microM calphostin C, blocked by 500 nM bisindolylmaleimide, and unaffected by the pseudosubstrate inhibitor PKC19-36. 4. Thus we find that two membrane-delimited signaling pathways that inhibit ion channels in rat SCG neurons are strongly attenuated by PMA, but signaling pathway(s) that use a diffusible cytoplasmic messenger are not. We speculate that a nonstandard PKC isoform, perhaps PKC mu, mediates PMA actions.
Shapiro
M S
MS
Department of Physiology and Biophysics, University of Washington, Seattle 98195-7290, USA.
Zhou
J
J
Hille
B
B
D000818:Animals; D015220:Calcium Channels / Q000187:drug effects*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000502:physiology*; D018721:Muscarinic Agonists / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology; D011493:Protein Kinase C / Q000187:drug effects; D011494:Protein Kinases / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D015220:Calcium Channels; D018721:Muscarinic Agonists; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate