Function and phylogeny support the independent evolution of acid-sensing ion channels in the Placozoa

Abstract

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.

Peripheral and central employment of acid-sensing ion channels during early bilaterian evolution

“…The clades most closely related to ASICs within the DEG/ENaC superfamily tree include three from which several channels have been characterized: mammalian bile acid-sensitive ion channels (BASICs), Trichoplax adhaerans Na + channels (TadNaCs); and HyNaC and Nematostella vectensis Na + channels (NeNaCs). We cannot establish phylogenetically the identity of the ancestral gene from which ASICs emerged, as branch support toward the base of the ASIC + BASIC + TadNaC + HyNaC/NeNaC clade is relatively low: our maximum likelihood (ML) trees inferred with aLRT SH-like and aBayes statistics yielded slightly different topologies within this clade ( Figure 2—figure supplement 1C and D ), and previous studies using similar methods to each other also inferred slightly different topologies within this clade ( Aguilar-Camacho et al, 2022 ; Elkhatib et al, 2022 ). Statistical support for the distinct ASIC clade is strong, however ( Figure 2—figure supplement 1 ).…”

“…HyNaCs are neuropeptide-gated channels from the medusozoan cnidarian Hydra ( Assmann et al, 2014 ), and when NeNaCs from the anthozoan cnidarian Nematostella were recently reported, surprisingly, NeNaC2 and NeNaC14 were activated by protons (pH 50 values of 5.8 and <4.0), not by cnidarian neuropeptides ( Aguilar-Camacho et al, 2022 ). The TadNaC clade also includes a proton-activated channel, TadNaC2 (pH 50 5.1), in addition to a proton-inhibited channel, TadNaC6 ( Elkhatib et al, 2019 ; Elkhatib et al, 2022 ). Thus, proton-activated and -inhibited channels occur sporadically throughout the ASIC + BASIC + TadNaC + HyNaC/NeNaC clade, and only in the ASIC clade is proton-induced activation the defining feature.…”

“…ASICs could thus have emerged from a proton-activated ancestor, after which this function was selected for; or from a peptide- or bile acid-activated ancestor, in which a novel mechanism of activation evolved. Certain functional and phylogenetic data have led others to favor the latter possibility ( Elkhatib et al, 2022 ; Golubovic et al, 2007 ), and indeed structural features important for channel gating throughout the prototypical ASIC family, such as highly conserved histidine residues and/or aromatic interactions, are absent from proton-gated NeNaCs and TadNaCs ( Elkhatib et al, 2022 ; Lynagh et al, 2018 ). The fact that proton-induced activation has emerged in numerous DEG/ENaC channels, even outside the ASIC + BASIC + TadNaC + HyNaC/NeNaC clade, as shown for a Drosophila PPK channel and three C. elegans degenerin-like channels ( Jang et al, 2019 ; Kaulich et al, 2022 ), suggests a propensity for this function in the DEG/ENaC superfamily.…”

Neuropeptides and degenerin/epithelial Na+ channels: a relationship from mammals to cnidarians

“…We will first consider these channels in bilaterian invertebrates before we turn to such channels in a cnidarian, the freshwater polyp Hydra magnipapillata. So far, no peptide-gated channels have been identified in deuterostome animals, including vertebrates, but many deuterostome DEG/ENaCs have not yet been deorphanized (Elkhatib et al, 2022), meaning that we cannot exclude their presence in deuterostomes.…”

Large-scale deorphanization ofNematostella vectensisneuropeptide GPCRs supports the independent expansion of bilaterian and cnidarian peptidergic systems

“…These include receptors for Hydra vulgaris RFamide peptides and a receptor for a PRXamide maturation-inducing hormone (MIH) in the hydrozoan Clytia hemisphaerica (Artigas et al, 2020; Assmann et al, 2014). Hydra RFamide peptides activate heterotrimeric peptide-gated ion channels belonging to the DEG/ENaC family and distantly related to bilaterian RFamide- and Wamide-gated ion channels (Dandamudi et al, 2022; Elkhatib et al, 2022; Gründer et al, 2022). The Clytia MIH receptor is a class A GPCR, which together with related cnidarian GPCRs shows a many-to-many ortholog relationship to a range of bilaterian neuropeptide GPCR families that also contain receptors for RFamide-like neuropeptides (Artigas et al, 2020).…”