ABSTRACT
ASIC channels are bilaterian proton-gated sodium channels belonging to the large and functionally-diverse Deg/ENaC family that also includes peptide- and mechanically-gated channels. Here, we report that the non-bilaterian invertebrate Trichoplax adhaerens possesses a proton-activated Deg/ENaC channel, TadNaC2, with a unique combination of biophysical features including tachyphylaxis like ASIC1a, reduced proton sensitivity like ASIC2a, biphasic macroscopic currents like ASIC3, as well as low sensitivity to the Deg/ENaC channel blocker amiloride and Ca2+ ions. Structural modeling and mutation analyses reveal that TadNaC2 proton gating is different from ASIC channels, lacking key molecular determinants, and involving unique residues within the palm and finger regions. Phylogenetic analysis reveals that a monophyletic clade of T. adhaerens Deg/ENaC channels, which includes TadNaC2, is phylogenetically distinct from ASIC channels, instead forming a clade with BASIC channels. Altogether, this work suggests that ASIC-like channels evolved independently in T. adhaerens and its phylum Placozoa. Our phylogenetic analysis also identifies several clades of uncharacterized metazoan Deg/ENaC channels, and provides phylogenetic evidence for the existence of Deg/ENaC channels outside of Metazoa, present in the gene data of select unicellular heterokont and filasterea-related species.
Subject(s)
Placozoa , Animals , Placozoa/genetics , Phylogeny , Protons , Acid Sensing Ion Channels/genetics , AmilorideABSTRACT
Neuropeptides are one of the largest and most diverse families of signaling molecules in animals and, accordingly, they regulate many physiological processes and behaviors. Genome and transcriptome sequencing has enabled the identification of genes encoding neuropeptide precursor proteins in species from a growing variety of taxa, including bilaterian and non-bilaterian animals. Of particular interest are deuterostome invertebrates such as the phylum Echinodermata, which occupies a phylogenetic position that has facilitated reconstruction of the evolution of neuropeptide signaling systems in Bilateria. However, our knowledge of neuropeptide signaling in echinoderms is largely based on bioinformatic and experimental analysis of eleutherozoans-Asterozoa (starfish and brittle stars) and Echinozoa (sea urchins and sea cucumbers). Little is known about neuropeptide signaling in crinoids (feather stars and sea lilies), which are a sister clade to the Eleutherozoa. Therefore, we have analyzed transcriptome/genome sequence data from three feather star species, Anneissia japonica, Antedon mediterranea, and Florometra serratissima, to produce the first comprehensive identification of neuropeptide precursors in crinoids. These include representatives of bilaterian neuropeptide precursor families and several predicted crinoid neuropeptide precursors. Using A. mediterranea as an experimental model, we have investigated the expression of selected neuropeptides in larvae (doliolaria), post-metamorphic pentacrinoids and adults, providing new insights into the cellular architecture of crinoid nervous systems. Thus, using mRNA in situ hybridization F-type SALMFamide precursor transcripts were revealed in a previously undescribed population of peptidergic cells located dorso-laterally in doliolaria. Furthermore, using immunohistochemistry a calcitonin-type neuropeptide was revealed in the aboral nerve center, circumoral nerve ring and oral tube feet in pentacrinoids and in the ectoneural and entoneural compartments of the nervous system in adults. Moreover, functional analysis of a vasopressin/oxytocin-type neuropeptide (crinotocin), which is expressed in the brachial nerve of the arms in A. mediterranea, revealed that this peptide causes a dose-dependent change in the mechanical behavior of arm preparations in vitro-the first reported biological action of a neuropeptide in a crinoid. In conclusion, our findings provide new perspectives on neuropeptide signaling in echinoderms and the foundations for further exploration of neuropeptide expression/function in crinoids as a sister clade to eleutherozoan echinoderms.
ABSTRACT
Neuropeptides are diverse signaling molecules in animals commonly acting through G-protein coupled receptors (GPCRs). Neuropeptides and their receptors underwent extensive diversification in bilaterians and the relationships of many peptide-receptor systems have been clarified. However, we lack a detailed picture of neuropeptide evolution in lophotrochozoans as in-depth studies only exist for mollusks and annelids. Here, we analyze peptidergic systems in Nemertea, Brachiopoda, and Phoronida. We screened transcriptomes from 13 nemertean, 6 brachiopod, and 4 phoronid species for proneuropeptides and neuropeptide GPCRs. With mass spectrometry from the nemertean Lineus longissimus, we validated several predicted peptides and identified novel ones. Molecular phylogeny combined with peptide-sequence and gene-structure comparisons allowed us to comprehensively map spiralian neuropeptide evolution. We found most mollusk and annelid peptidergic systems also in nemerteans, brachiopods, and phoronids. We uncovered previously hidden relationships including the orthologies of spiralian CCWamides to arthropod agatoxin-like peptides and of mollusk APGWamides to RGWamides from annelids, with ortholog systems in nemerteans, brachiopods, and phoronids. We found that pleurin neuropeptides previously only found in mollusks are also present in nemerteans and brachiopods. We also identified cases of gene family duplications and losses. These include a protostome-specific expansion of RFamide/Wamide signaling, a spiralian expansion of GnRH-related peptides, and duplications of vasopressin/oxytocin before the divergence of brachiopods, phoronids, and nemerteans. This analysis expands our knowledge of peptidergic signaling in spiralians and other protostomes. Our annotated data set of nearly 1,300 proneuropeptide sequences and 600 GPCRs presents a useful resource for further studies of neuropeptide signaling.
