CAMP/PKA Pathways and S56 Phosphorylation Are Involved in AA/PGE2-Induced Increases in rNaV1.4 Current.

Hua Gu,Ping Chen,Yan-Jia Fang,Dong-Dong Liu,Yan-Ai Mei,Steven Barnes

doi:10.1371/journal.pone.0140715

Abstract

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 rNaV1.4 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 rNaV1.4 current peak without modulating the steady-state activation and inactivation properties of the rNaV1.4 channel. These results differed from the effects of extracellular application of AA on rNaV1.4 current. The effects of intracellular AA were mimicked by prostaglandin E2 but not eicosatetraynoic acid (ETYA), the non-metabolized analogue of AA, and were eliminated by treatment with cyclooxygenase inhibitors, flufenamic acid, or indomethacin. AA/PGE2-induced activation of rNaV1.4 channels was mimicked by a cAMP analogue (db-cAMP) and eliminated by a PKA inhibitor, PKAi. Furthermore, inhibition of EP2 and EP4 (PGE2 receptors) with AH6809 and AH23848 reduced the intracellular AA/PGE2-induced increase of rNaV1.4 current. Two mutated channels, rNaV1.4S56A and rNaV1.4T21A, were designed to investigate the role of predicted phosphorylation sites in the AA/PGE2–mediated regulation of rNaV1.4 currents. In rNaV1.4S56A, the effects of intracellular db-cAMP, AA, and PGE2 were significantly reduced. The results of the present study suggest that intracellular AA augments rNaV1.4 current by PGE2/EP receptor-mediated activation of the cAMP/PKA pathway, and that the S56 residue on the channel protein is important for this process.

Highlights

Arachidonic acid (AA) is a polyunsaturated fatty acid cleaved from cell membrane phospholipid molecules via the action of the enzyme PLA2
Our studies indicate that intracellular AA activates rNaV1.4 currents via the AA!prostaglandin E2 (PGE2)!EP receptor!cAMP/PKA pathway, and that the PKA phosphorylation site at S56 on the channel protein may be involved in the activity of AA
These results suggest that the cAMP/PKA pathway is associated with the AA/PGE2induced increase of the rNaV1.4 current

Summary

Introduction

Arachidonic acid (AA) is a polyunsaturated fatty acid cleaved from cell membrane phospholipid molecules via the action of the enzyme PLA2. AA is a biologically active signaling molecule that plays important roles in neurons and muscle under both physiological and pathological conditions [1, 2]. Its effects include modulation of the activity of protein kinases, elevation of intracellular Ca2+ levels, and regulation of neuronal excitability [3,4,5]. Regulation of ion channel activity by AA may occur via direct effects, where AA interacts directly with ion channel proteins, or through perturbation of the plasma membrane [7, 10, 11]. Free intracellular AA serves as a key transient cell signaling intermediate and undergoes rapid enzymatic conversion to diverse metabolites, including prostaglandins (PGs, such as PGD2, PGE2 and thromboxane A2) and the leukotriene/lipoxin (LX) families of eicosanoids [13].

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