cAMP/PKA Pathways and S56 Phosphorylation Are Involved in AA/PGE(2)-Induced Increases in rNa(V)1.4 Current

Arachidonic acid (AA) and its metabolites are important second messengers for ion channel modulation. The effects of extracellular application of AA and its non-metabolized analogue on muscle rNa(V)1.4 Na(+) current has been studied, but little is known about the effects of intracellular application of AA on this channel isoform. Here, we report that intracellular application of AA significantly augmented the rNa(V)1.4 current peak without modulating the steady-state activation and inactivation properties of the rNa(V)1.4 channel. These results differed from the effects of extracellular application of AA on rNa(V)1.4 current. The effects of intracellular AA were mimicked by prostaglandin E(2) but not eicosatetraynoic acid (ETYA), the non-metabolized analogue of AA, and were eliminated by treatment with cyclooxygenase inhibitors, flufenamic acid, or indomethacin. AA/PGE(2)-induced activation of rNa(V)1.4 channels was mimicked by a cAMP analogue (db-cAMP) and eliminated by a PKA inhibitor, PKAi. Furthermore, inhibition of EP2 and EP4 (PGE(2) receptors) with AH6809 and AH23848 reduced the intracellular AA/PGE(2)-induced increase of rNa(V)1.4 current. Two mutated channels, rNa(V)1.4S56A and rNa(V)1.4T21A, were designed to investigate the role of predicted phosphorylation sites in the AA/PGE(2)–mediated regulation of rNa(V)1.4 currents. In rNa(V)1.4S56A, the effects of intracellular db-cAMP, AA, and PGE(2) were significantly reduced. The results of the present study suggest that intracellular AA augments rNa(V)1.4 current by PGE(2)/EP receptor-mediated activation of the cAMP/PKA pathway, and that the S56 residue on the channel protein is important for this process.