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 alpha, beta,or gamma C terminus were not inhibited by arachidonic acid or ETYA. Furthermore, mutation of Tyr618 in the PY motif of the beta 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 beta subunit C-terminal truncations revealed a second region N-terminal to the PY motif (spanning residues betaVal580-betaGly599) 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.
Highlights
Epithelial Naϩ channels (ENaCs)1 are expressed in apical plasma membranes of principal cells in the distal nephron, airway, and alveolar epithelia in the lung, surface cells in the distal colon, urinary bladder epithelia, and other tissues including ducts of salivary and sweat glands
Channels with a truncated ␣, , or ␥ C terminus were not inhibited by arachidonic acid or Eicosatetraynoic acid (ETYA)
Analyses of channels expressing a series of  subunit C-terminal truncations revealed a second region N-terminal to the PY motif that allowed for ETYA-mediated ENaC inhibition
Summary
Epithelial Naϩ channels (ENaCs) are expressed in apical plasma membranes of principal cells in the distal nephron, airway, and alveolar epithelia in the lung, surface cells in the distal colon, urinary bladder epithelia, and other tissues including ducts of salivary and sweat glands. The three subunits are similar in overall structure, with intracellular N and C termini, two transmembrane spanning domains, and a large extracellular domain Several hormones, such as aldosterone, arginine vasopressin, insulin, as well as selected kinases, modulate ENaC activity. The 85-kDa cPLA2 is a ubiquitously expressed enzyme and is the only PLA2 that preferentially hydrolyzes the sn-2 position of phospholipids to produce arachidonic acid. The epithelial cell line A6 derived from Xenopus laevis kidney, grown on non-permeable supports, expressed apical Naϩ channels that were regulated by the activities of phospholipase and lipoxygenase enzymes (13). A recent study reported that inhibition of PLA2 by the addition of aristolochic acid to the apical bath increased amiloride-sensitive transepithelial Naϩ transport in A6 cells and was associated with a reduction in the production of arachidonic acid. This decrease in Naϩ transport was reversed by prostaglandin E2 (14, 15)
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