8-pCPT-cGMP stimulates αβγ-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties

Abstract

Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of alphabetagamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alphabetagamma-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat alphabetagamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alphabetagamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N(2)-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH(2)- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human alphabetagamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the para-chlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na+ Self-Inhibition of Epithelial Na+ Channels

“…Our previous studies found that heterologous ENaC expressed in oocytes may not be regulated by PKG (19). In PKG-deficient cells, the extent of compound stimulation of ENaC was identical to those transfected with scramble siRNA.…”

“…CPT-cGMP, but not parent cGMP, acutely activated human ENaC activity as an external ligand (19,26). The aim of this study was to examine whether CPT-cGMP could be used as a pharmaceutical strategy to up-regulate alveolar fluid clearance (AFC) via stimulating ENaC and its underlying mechanisms.…”

“…Among these, halothane and Cl 2 ions may activate ENaC by eliminating Na 1 self-inhibition (17,18). We recently found that 8-(4-chlorophenylthio)-guanosine-39,59-cyclic monophosphate-Na (CPT-cGMP) stimulated human abg ENaC in oocytes in a PKG-independent and domain-specific manner (19). The underlying mechanisms are unknown.…”

“…A mutation (a F61L ) at the amino terminal of the protein, adjacent to the gating tract in the M1 domain, enhanced self-inhibition (25). Our current understanding of the nature of ENaC gating mechanisms provides no unequivocal answers to the question of how the aforementioned domains affect self-inhibition.CPT-cGMP, but not parent cGMP, acutely activated human ENaC activity as an external ligand (19,26). The aim of this study was to examine whether CPT-cGMP could be used as a pharmaceutical strategy to up-regulate alveolar fluid clearance (AFC) via stimulating ENaC and its underlying mechanisms.…”

External Cu2+ Inhibits Human Epithelial Na+ Channels by Binding at a Subunit Interface of Extracellular Domains

“…External amiloride analogs, cations, anions, nucleotides, serine proteases, and laminar shear stress inhibit or stimulate endogenous or exogenous ENaCs (5)(6)(7)(8)(9)(10)(11)(12). All of these extracellular regulators appear to directly alter the activity of ENaCs in plasma membranes rather than affect channel subunit trafficking (1).…”