Roles of G-protein beta gamma, arachidonic acid, and phosphorylation inconvergent activation of an S-like potassium conductance by dopamine, Ala-Pro-Gly-Trp-NH2, and Phe-Met-Arg-Phe-NH2.
Dopamine and the neuropeptides Ala-Pro-Gly-Trp-NH2 (APGWamide or APGWa) and Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) all activate an S-like potassium channel in the light green cells of the mollusc Lymnaea stagnalis, neuroendocrine cells that release insulin-related peptides. We studied the signaling pathways underlying the responses, the role of the G-protein betagamma subunit, and the interference by phosphorylation pathways. All responses are blocked by an inhibitor of arachidonic acid (AA) release, 4-bromophenacylbromide, and by inhibitors of lipoxygenases (nordihydroguaiaretic acid and AA-861) but not by indomethacin, a cyclooxygenase inhibitor. AA and phospholipase A2 (PLA2) induced currents with similar I-V characteristics and potassium selectivity as dopamine, APGWa, and FMRFa. PLA2 occluded the response to FMRFa. We conclude that convergence of the actions of dopamine, APGWa, and FMRFa onto the S-like channel occurs at or upstream of the level of AA and that formation of lipoxygenase metabolites of AA is necessary to activate the channel. Injection of a synthetic peptide, which interferes with G-protein betagamma subunits, inhibited the agonist-induced potassium current. This suggests that betagamma subunits mediate the response, possibly by directly coupling to a phospholipase. Finally, the responses to dopamine, APGWa, and FMRFa were inhibited by activation of PKA and PKC, suggesting that the responses are counteracted by PKA- and PKC-dependent phosphorylation. The PLA2-activated potassium current was inhibited by 8-chlorophenylthio-cAMP but not by 12-O-tetradecanoylphorbol 13-acetate (TPA). However, TPA did inhibit the potassium current induced by irreversible activation of the G-protein using GTP-gamma-S. Thus, it appears that PKA targets a site downstream of AA formation, e.g., the potassium channel, whereas PKC acts at the active G-protein or the phospholipase.
van Tol-Steye
H
H
Department of Neurophysiology, Research Institute Neurosciences, Faculty of Chemistry, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands.
Lodder
J C
JC
Mansvelder
H D
HD
Planta
R J
RJ
van Heerikhuizen
H
H
Kits
K S
KS
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D004298:Dopamine / Q000494:pharmacology*; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019835:FMRFamide / Q000494:pharmacology; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins; D008195:Lymnaea; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000494:pharmacology; D010766:Phosphorylation; D015221:Potassium Channels / Q000819:agonists*; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D009479:Neuropeptides; D015221:Potassium Channels; C063193:alanyl-prolyl-glycyl-tryptophanamide; D016718:Arachidonic Acid; D019835:FMRFamide; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D004298:Dopamine