Currie, M. A. | bioRxiv Science

bioRxiv2024

The binding of Mint/X11 PDZ domains to CaV2 calcium channels predates bilaterian animals

PDZ domain mediated interactions with voltage-gated calcium (CaV) channel C-termini play important roles in localizing membrane Ca2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and CaV2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to CaV2 channels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and CaV2 channels from cnidarians and placozoans interact in vitro, and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis. Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly binds the divergent C-terminal ligands of cnidarian and placozoan CaV2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore CaV2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind CaV2 channels predates pre-bilaterian animals, and that evolutionary changes in CaV2 channel C-terminal sequences resulted in altered binding modalities with Mint.

evolutionary biology↗

bioRxiv2022

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

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.

evolutionary biology↗