Ile481 from the Guinea-Pig α-subunit Plays a Major Role in the Activation of ENaC by cpt-cAMP

Abstract

The epithelial sodium channel (ENaC) is the major rate-limiting step for vasopressin and aldosterone sensitive Na⁺ reabsorption across kidney epithelia. Recently, ENaC activity was shown to be modulated by extracellular factors such as proteases, Na⁺ ion and several other elements. However, the molecular mechanisms of these actions remain unclear. We and others have shown that ENaC composed of the guinea-pig α-subunit (αgp), and the β γ rat subunits (βrγr) could be activated by cpt-cAMP, a cAMP analogue, through a mechanism not involving the cAMP-PKA pathway. In the present study, we confirmed by patch-clamp experiments on Xenopus oocytes that the number of open channels increased by 2.4-fold after cpt-cAMP exposure. In order to characterize the extracellular domain involved in this activation, we generated α-subunit chimera's harboring different portions of the extracellular loop of the αgp and αr. Using two-electrode voltage-clamp, we established that Tyr456-Ser532 from the αgp confers sensibility to cpt-AMP. Then, by site-directed mutagenesis, we have isolated Ile481 as a major residue for cpt-cAMP-dependant activation. Taken together, these experiments provide evidence of an extracellular-ligand stimulating ENaC. They also contribute to the further understanding of the structure-function relationship of this channel.

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

“…Indeed, 8-CPT-cAMP also activated ENaC activity. These observations are in good agreement with previous reports (7,35).…”

“…In sharp contrast to the native cAMP-activated ENaC, heterologously expressed ␣␤␥-ENaC in oocytes has been considered to be cAMP/PKA independent, particularly for acute regulation (55). Furthermore, a critical extracellular domain has recently been identified as 8-CPTcAMP binding site (35), indicating that 8-CPT-cAMP is an extracellular ligand for ENaC (18,35). Combined with our similar diverse observations for the activation of native and cloned ENaC by 8-pCPT-cGMP, 8-pCPT-cGMP may serve as a new ENaC ligand to regulate channel activity extracellularly.…”

“…To answer this question, we used cell-free outside-out mode to exclude the involvement of soluable cytosolic signal molecules. 8-CPT-cAMP has been recently confirmed as a ligand to intermolecularly regulate the extracellular domains of cloned ENaC in oocytes (18,35). As a positive control (Fig.…”

“…8-pCPT-cGMP may activate ENaC as an external ligand, as several luminal reagents, for example, amiloride, protons, and zinc ions, have all been considered to regulate ENaC by interacting with ectoloop of the channel proteins (18). On the other hand, it has been proposed that 8-CPT-cAMP and 8-pCPT-cGMP may activate ENaC and cystic fibrosis transmembrane conductance regulator, respectively, not via the widely accepted cAMP/ PKA and cGMP/PKG pathways (35,47). If this is also a scenario for 8-pCPT-cGMP to activate ENaC, a defined cGMP structure is required.…”

New Insights into the Epithelial Sodium Channel Using Directed Mutagenesis

“…The N510I mutation in the gene for the rat subunit caused intermediate sensitivity to cpt-cAMP. All other mutations or combination of mutations, including N510I in the rat gene, did not increase the cpt-cAMP effect (Renauld et al, 2008).…”