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.

Background Trichoplax adhaerens is a fascinating early-diverging animal that lacks a nervous system and synapses, and yet is capable of directed motile feeding behavior culminating in the external digestion of microorganisms by secreted hydrolytic enzymes. The mechanisms by which Trichoplax cells communicate with each other to coordinate their activity and behavior is unclear, though recent studies have suggested that secreted regulatory peptides might be involved.Results Here, we generated a high quality mRNA transcriptome of Trichoplax adhaerens , and predicted secreted proteins to identify gene homologues for digestion, development, immunity, cell adhesion, and peptide signaling. Detailed annotation of the expressed Trichoplax gene set also identified a nearly complete set of electrogenic genes involved in fast neural signalling, plus a set of 665 G-protein coupled receptors that in the nervous system integrate with fast signalling machinery to modulate cellular excitability. Furthermore, Trichoplax expresses an array of genes involved in intracellular signaling, including the key effector enzymes protein kinases A and C that functionally link fast and slow cellular signaling. Also identified were nearly complete sets of pre- and post-synaptic scaffolding genes, most encoding appropriate protein domain architectures. Notably, the Trichoplax proteome was found to bear slightly reduced counts of synaptic protein interaction domains such as PDZ, SH3 and C2 compared to other animals, but abundance of these domains did not appear to predict the presence of synapses in early-diverging groups.Conclusions Despite its apparent cellular and morphological simplicity, Trichoplax expresses a rich set of genes involved in complex animal traits. The transcriptome presented here adds a valuable additional resource for molecular studies on Trichoplax genes, exemplified by our ability to clone cDNAs for nine full-length acid sensing ion channel proteins with almost perfect matches with their corresponding transcriptome sequences.

The dominant role of CaV2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of CaV2 and CaV1 channels, and less so CaV3 channels, it is unclear why there have not been major shifts towards dependency on other CaV channels for synaptic transmission. Here, we provide a structural and functional profile of the CaV2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possess single gene homologues for CaV1-CaV3 channels. Remarkably, the highly divergent channel possesses similar features as human CaV2.1 and other CaV2 channels, including high voltage-activated currents that are larger in external Ba2+ than Ca2+, voltage dependent kinetics of activation, inactivation and deactivation, and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax CaV2 suggests that the core features of pre-synaptic CaV2 channels were established early during animal evolution, after CaV1 and CaV2 channels emerged via proposed gene duplication from an ancestral CaV1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian CaV2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA, and to metal cation blockers Cd2+ and Ni2+. Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gβγ subunits, which nevertheless inhibited the human CaV2.1 channel suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G-proteins. Lastly, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior, and other likely secretory cells, suggesting contributions to regulated exocytosis.

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Acid-sensing ion channels (ASICs) are proton-gated cation channels that are part of the Deg/ENaC ion channel family, which also includes neuropeptide-, bile acid-, and mechanically-gated channels. Despite sharing common tertiary and quaternary structures, strong sequence divergence within the Deg/ENaC family has made it difficult to resolve their phylogenetic relationships, and by extension, whether channels with common functional features, such as proton-activation, share common ancestry or evolved independently. Here, we report that a Deg/ENaC channel from the early diverging placozoan species Trichoplax adhaerens, named TadNaC2, conducts proton-activated currents in vitro with biophysical features that resemble those of the mammalian ASIC1 to ASIC3 channels. Through a combined cluster-based and phylogenetic analysis, we successfully resolve the evolutionary relationships of most major lineages of metazoan Deg/ENaC channels, identifying two subfamilies within the larger Deg/ENaC family that are of ancient, pre-bilaterian origin. We also identify bona fide Deg/ENaC channel homologues from filasterean and heterokont single celled eukaryotes. Furthermore, we find that ASIC channels, TadNaC2, and various other proton-activated channels from vertebrates and invertebrates are part of phylogenetically distinct lineages. Through structural modelling and mutation analysis, we find that TadNaC2 proton-activation employs fundamentally different molecular determinants than ASIC channels, and identify two unique histidine residues in the placozoan channel that are required for its proton-activation. Together, our phylogenetic and functional analyses support the independent evolution of proton-activated channels in the phylum Placozoa. Spurred by our discovery of pH sensitive channels, we discovered that despite lacking a nervous system, Trichoplax can sense changes in extracellular pH to coordinate its various cell types to locomote away from acidic environments, and to contract upon rapid exposure to acidic pH in a Ca2+-dependent manner. Lastly, via yeast 2 hybrid screening, we find that the Trichoplax channels TadNaC2 and TadNaC10, belonging to the two separate Deg/ENaC subfamilies, interact with the cytoskeleton organizing protein filamin, similar to the interaction reported for the human ENaC channels.