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 Ca(2+) ions modulate gating, and in some cases, the binding of protons and Ca(2+) 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 Ca(2+) We determine that proton block is enhanced in low external Ca(2+) 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 Ca(2+) sensitivity in other DEG/ENaC channels, and a mutation of one conserved residue (S353A) associated with Ca(2+) block in rodent BASIC channels instead affected proton sensitivity, all indicative of independent evolution of H(+) and Ca(2+) 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, Ca(2+), and amiloride regulation of DEG/ENaC channels and insight into the possible core-gating features of ancestral ion channels.