Abstract

The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution. The epithelial sodium channel (ENaC) is present in the apical membrane of sodium-absorbing vertebrate epithelia and evolved as part of a machinery for efficient sodium conservation. ENaC belongs to the degenerin/ENaC protein family and is the only member that opens without an external stimulus. We hypothesized that ENaC evolved from a proton-activated sodium channel present in ionocytes of freshwater vertebrates and therefore investigated whether such ancestral traits are present in ENaC isoforms of the aquatic pipid frog Xenopus laevis Using whole-cell and single-channel electrophysiology of Xenopus oocytes expressing ENaC isoforms assembled from αβγ- or δβγ-subunit combinations, we demonstrate that Xenopus δβγ-ENaC is profoundly activated by extracellular acidification within biologically relevant ranges (pH 8.0-6.0). This effect was not observed in Xenopus αβγ-ENaC or human ENaC orthologs. We show that protons interfere with allosteric ENaC inhibition by extracellular sodium ions, thereby increasing the probability of channel opening. Using homology modeling of ENaC structure and site-directed mutagenesis, we identified a cleft region within the extracellular loop of the δ-subunit that contains several acidic amino acid residues that confer proton-sensitivity and enable allosteric inhibition by extracellular sodium ions. We propose that Xenopus δβγ-ENaC can serve as a model for investigating ENaC transformation from a proton-activated toward a constitutively-active ion channel. Such transformation might have occurred during the evolution of tetrapod vertebrates to enable bulk sodium absorption during the water-to-land transition.

Highlights

  • The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution

  • Using whole-cell and single-channel electrophysiology of Xenopus oocytes expressing ENaC isoforms assembled from ␣␤␥- or ␦␤␥-subunit combinations, we demonstrate that Xenopus ␦␤␥-ENaC is profoundly activated by extracellular acidification within biologically relevant ranges

  • Current levels normalized to the initial peak in IM followed a twophase decay function (Fig. 1b), where 39.6 Ϯ 3.1% (n ϭ 14) of the total current decline was attributable to the initial fraction of IM decay (Fig. 1c)

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Summary

Introduction

The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution. We propose that Xenopus ␦␤␥-ENaC can serve as a model for investigating ENaC transformation from a proton-activated toward a constitutivelyactive ion channel Such transformation might have occurred during the evolution of tetrapod vertebrates to enable bulk sodium absorption during the water–to–land transition. A recent study resolving the structure of human ␣␤␥-ENaC revealed that each subunit contains short intracellular N and C termini that are connected by two transmembrane helices and a large extracellular domain [7]. The topology of this ectodomain resembles a clenched hand comprising the “palm,” “knuckle,”

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