A Na+ leak channel cloned from Trichoplax adhaerens extends extracellular pH and Ca2+ sensing for the DEG/ENaC family close to the base of Metazoa

Wassim Elkhatib,Carolyn L Smith,Adriano Senatore

doi:10.1074/jbc.ra119.010542

Abstract

Acid-sensitive ion channels belonging to the degenerin/epithelial sodium channel (DEG/ENaC) family activate in response to extracellular protons and are considered unique to deuterostomes. However, sensitivity to pH/protons is more widespread, where, for example, human ENaC Na+ leak channels are potentiated and mouse BASIC and Caenorhabditis elegans ACD-1 Na+ leak channels are blocked by extracellular protons. For many DEG/ENaC channels, extracellular Ca2+ ions modulate gating, and in some cases, the binding of protons and Ca2+ is interdependent. Here, we functionally characterize a DEG/ENaC channel from the early-diverging animal Trichoplax adhaerens, TadNaC6, that conducts Na+-selective leak currents in vitro sensitive to blockade by both extracellular protons and Ca2+. We determine that proton block is enhanced in low external Ca2+ concentration, whereas calcium block is enhanced in low external proton concentration, indicative of competitive binding of these two ligands to extracellular sites of the channel protein. TadNaC6 lacks most determinant residues for proton and Ca2+ sensitivity in other DEG/ENaC channels, and a mutation of one conserved residue (S353A) associated with Ca2+ block in rodent BASIC channels instead affected proton sensitivity, all indicative of independent evolution of H+ and Ca2+ sensitivity. Strikingly, TadNaC6 was potently activated by the general DEG/ENaC channel blocker amiloride, a rare feature only reported for the acid-activated channel ASIC3. The sequence and structural divergence of TadNaC6, coupled with its noncanonical functional features, provide unique opportunities for probing the proton, Ca2+, and amiloride regulation of DEG/ENaC channels and insight into the possible core-gating features of ancestral ion channels.

Highlights

Acid-sensitive ion channels belonging to the degenerin/epithelial sodium channel (DEG/ENaC) family activate in response to extracellular protons and are considered unique to deuterostomes
TadNaC6 was found to be activated by the general DEG/ENaC channel blocker amiloride, a rare feature only reported for rat ASIC3 [23] and a mutant version of human ASIC2 [24], but lacks key residues attributed to this phenomenon
No protostome invertebrate DEG/ENaC channels clustered between the Trichoplax and vertebrate Acid-sensitive ion channels (ASICs) clades, and generally, the phylogenetic positions of the various ion channels throughout the tree do not correspond with the metazoan phylogeny (Fig. 1A, inset)

Summary

To whom correspondence should be addressed

In vitro properties of a placozoan DEG/ENaC ion channel better understanding about the core characteristics and evolution of this large family of ion channels. In vitro expression of TadNaC6 in Chinese hamster ovary (CHO) cells, coupled with whole-cell patch-clamp recording, revealed robust Naϩ-selective leak currents that were blocked by both external protons and Ca2ϩ, a functional profile reported for the mouse BASIC and nematode ACD-1 DEG/ENaC channels [5, 11]. Given its phylogenetic distance from the functionally similar mouse BASIC and nematode ACD-1 Naϩ leak channels, and the absence of key amino acids involved in proton and Ca2ϩ sensitivity in other DEG/ENaC channels, it is likely that the gating features of TadNaC6 evolved independently. TadNaC6 was found to be activated by the general DEG/ENaC channel blocker amiloride, a rare feature only reported for rat ASIC3 [23] and a mutant version of human ASIC2 [24], but lacks key residues attributed to this phenomenon. The highly-divergent sequence and atypical properties observed for TadNaC6 provide some unique perspectives on the structure, function, and evolution of proton- and Ca2ϩ-sensitive DEG/ENaC channels

Results

Discussion

Experimental procedures