A Na leak channel cloned from extends extracellular pH and Ca sensing for the DEG/ENaC family close to the base of Metazoa.

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 ACD-1 Na leak channels are blocked by extracellular protons. For many DEG/ENaC channels, extracellular Ca ions modulate gating, and in some cases, the binding of protons and Ca is interdependent. Here, we functionally characterize a DEG/ENaC channel from the early-diverging animal , NaC6, that conducts Na-selective leak currents sensitive to blockade by both extracellular protons and Ca We determine that proton block is enhanced in low external Ca 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. NaC6 lacks most determinant residues for proton and Ca sensitivity in other DEG/ENaC channels, and a mutation of one conserved residue (S353A) associated with Ca block in rodent BASIC channels instead affected proton sensitivity, all indicative of independent evolution of H and Ca sensitivity. Strikingly, NaC6 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 NaC6, coupled with its noncanonical functional features, provide unique opportunities for probing the proton, Ca, and amiloride regulation of DEG/ENaC channels and insight into the possible core-gating features of ancestral ion channels.