Roles of G-protein βγ, arachidonic acid, and phosphorylation in convergent activation of an S-like potassium conductance by dopamine, Ala- Pro-Gly-Trp-NH2, and Phe-Met-Arg-Phe-NH2

Abstract

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 βγ, 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 (PEA2) 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 βγ, subunits, inhibited the agonist-induced potassium current. This suggests that βγ, 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-γ-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.