Arachidonic acid regulates surface expression of epithelial sodium channels

Abstract

Epithelial Na+ channels (ENaCs) are regulated by the phospholipase A2 (PLA2) product arachidonic acid. Pharmacological inhibition of PLA2 with aristolochic acid induced a significant increase in amiloride-sensitive currents in Xenopus oocytes expressing ENaC. Arachidonic acid or 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolized analog of arachidonic acid, induced a time-dependent inhibition of Na+ transport. These effects were also observed by co-expression of a calcium-independent or a calcium-dependent PLA2. Channels with a truncated α, β, or γ C terminus were not inhibited by arachidonic acid or ETYA. Furthermore, mutation of Tyr618 in the PY motif of the β subunit abrogated the inhibitory effect of ETYA, suggesting that intact PY motifs participate in arachidonic acid-mediated ENaC inhibition. Analyses of channels expressing a series of β subunit C-terminal truncations revealed a second region N-terminal to the PY motif (spanning residues βVal580-βGly599) that allowed for ETYA-mediated ENaC inhibition. Analyses of both ENaC surface expression and ENaC trafficking with mutants that either gate channels open or closed in response to [(2-(trimethylammonium) ethyl] methanethiosulfonate bromide, or with brefeldin A, suggest that ETYA reduces channel surface expression by inhibiting ENaC exocytosis and increasing ENaC endocytosis.