(614.10) Activation by Cleavage of the Epithelial Na+ Channel α and γ Subunits Independently Coevolved with the Vertebrate Terrestrial Migration

Poster Board Number: E585

Ossama Kashlan (University of Pittsburgh), Xue-Ping Wang (University of Pittsburgh), Deidra Balchak (University of Pittsburgh), Clayton Gentilcore (University of Pittsburgh), Nathan Clark (University of Utah)

"Vertebrates evolved mechanisms for sodium conservation and gas exchange in conjunction with migration from aquatic to terrestrial habitats. Epithelial Na+ channel (ENaC) function is critical to systems responsible for extracellular fluid homeostasis and gas exchange. ENaC is activated by cleavage at multiple specific extracellular polybasic sites, releasing inhibitory tracts from the channel’s α and γ subunits. Here we investigated the evolution of ENaC regulatory mechanisms to determine which features coevolved with the marine-terrestrial transition. We consistently found both activating cleavage sites in the ENaC α and γ subunits of terrestrial vertebrates, while they appeared only sporadically in fishes. We confirmed that cleavage occurred at sites found in the γ subunit from Australian lungfish, leading to channel activation. Phylogenetic analysis and likelihood ratio tests showed that proximal and distal polybasic tracts in ENaC subunits coevolved, consistent with the dual cleavage requirement for activation. They also showed a coevolutionary dependence of tandem polybasic tracts with terrestrial status and with lungs, coincident with the ENaC activator aldosterone. Amplification of transcripts by RT-PCR in ray-finned Polypteriformes and previously in lobe-finned lungfish suggest that transcripts for ENaC subunits with cleavage sites are readily detected at important sites of ion exchange (gills and kidney), but are difficult to detect in lungs. Analysis of ancestral reconstructions strongly suggests that the polybasic tracts appeared independently in the α and γ subunits of ENaC. Similar analyses of the PY motif, required for Nedd4-2 dependent regulation, showed no coevolutionary pattern and that the PY motif first arose in an ancient ancestral ENaC subunit. Our data suggest that changes associated with adaptation to terrestrial life provided selective pressure for the development of ENaC activation by proteolytic cleavage.

This work was supported by NIDDK, National Institutes of Health, Grant R01 DK125439 (to O.B.K). The Pittsburgh Center for Kidney Research was supported by P30DK079307 from NIDDK, and the Pittsburgh Liver Research Center was supported by P30DK120531 from NIDDK.



Evolution of aldosterone signaling (MR, 11β-HSD2, and aldosterone synthase), air-breathing organs, ENaC, and ENaC regulatory motifs. ENaC subunits were absent in non-vertebrates and teleosts. Ancestral ENaC likely had a PY motif and was regulated by Nedd4-2. Dual ENaC cleavage sites appeared sporadically in marine species, and consistently in the ENaC α and γ subunits of tetrapods. CR, corticoid receptor; MR, mineralocorticoid receptor; GR, glucocorticoid receptor."