The binding of Mint/X11 PDZ domains to Ca V 2 calcium channels predates bilaterian animals.

PDZ domain mediated interactions with voltage-gated calcium (Ca V ) channel C-termini play important roles in localizing and compartmentalizing membrane Ca 2+ signaling. The first such interaction discovered was between the neuronal multi-domain protein Mint-1, and the presynaptc calcium channel Ca V 2.2 in mammals. Although the physiological significance of this interaction is unclear, its occurrence in vertebrates and bilaterian invertebrates suggests important and conserved functions. In this study, we explore the evolutionary origins of Mint and its interaction with Ca V 2 channels. Phylogenetic and structural in silico analyses revealed that Mint is an animal-specific gene, like Ca V 2 channels, which bears a highly divergent N-terminus but 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. Also deeply conserved are other Mint interacting proteins, namely amyloid precursor and related proteins, presenilins, neurexin, as well as CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast 2-hybrid and bacterial 2-hybrid experiments, we show that Mint and Ca V 2 channels from cnidarians and placozoans interact in vitro , and in situ hybridization revealed co-expression of corresponding transcripts in dissociated neurons from the cnidarian Nematostella vectensis . Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis was able to strongly bind the divergent C-terminal ligands of cnidarian and placozoan Ca V 2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore Ca V 2 channel C-terminus. Altogether, our analyses provide a model for the emergence of this interaction in early animals first via adoption of a PDZ ligand by Ca V 2 channels, followed by sequence changes in the ligand that caused a modality switch for binding to Mint.

Function and phylogeny support the independent evolution of an ASIC-like Deg/ENaC channel in the Placozoa.

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.

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria.

CaV1 and CaV2 voltage-gated calcium channels evolved from an ancestral CaV1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian CaV1 channels are uniquely subject to pronounced, buffer-resistant Ca2+/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca2+ rises. Early diverging, nonbilaterian invertebrates also possess CaV1 and CaV2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both CaV1 and CaV2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of CaV1 and CaV2 channels. Here, we build upon previous characterization of Trichoplax CaV channels by determining the cellular expression and ion-conducting properties of the CaV1 channel orthologue, TCaV1. We show that TCaV1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage-activated Ca2+ currents with kinetics resembling those of rat CaV1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCaV1, but not TCaV2, exhibits buffer-resistant Ca2+/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca2+ signaling within various cellular contexts.

Functional Studies of Trichoplax adhaerens Voltage-Gated Calcium Channel Activity.

Trichoplax adhaerens is a member of the phylum Placozoa, an enigmatic group of benthic animals with remarkably simple morphology. While initial work on these organisms has primarily focused on their morphology and the development of genomic resources, Trichoplax has received increased attention as a model for studying the evolution of nervous and sensory systems. This work is motivated by the fact that Trichoplax features distinct behaviours and responses to environmental stimuli. Therefore, much progress has been made in recent years on the molecular, cellular, and behavioral understanding of this organism. Methods outlined here provide hands-on approaches to cutting edge molecular and cellular techniques to record cellular activities in Trichoplax.

Conserved biophysical features of the CaV2 presynaptic Ca2+ channel homologue from the early-diverging animal Trichoplax adhaerens.

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 toward dependence 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 possesses 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 in 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 presynaptic 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 Gßγ 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. Last, 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.

Early Metazoan Origin and Multiple Losses of a Novel Clade of RIM Presynaptic Calcium Channel Scaffolding Protein Homologs.

The precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which, Rab3-interacting molecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channels in vitro via a PDZ domain that binds to the extreme C-termini of the channels at acidic ligand motifs of D/E-D/E/H-WC-COOH, and knockout of RIM in vertebrates and invertebrates disrupts CaV2 channel synaptic localization and synapse function. Here, we describe a previously uncharacterized clade of RIM proteins bearing domain architectures homologous to those of known RIM homologs, but with some notable differences including key amino acids associated with PDZ domain ligand specificity. This novel RIM emerged near the stem lineage of metazoans and underwent extensive losses, but is retained in select animals including the early-diverging placozoan Trichoplax adhaerens, and molluscs. RNA expression and localization studies in Trichoplax and the mollusc snail Lymnaea stagnalis indicate differential regional/tissue type expression, but overlapping expression in single isolated neurons from Lymnaea. Ctenophores, the most early-diverging animals with synapses, are unique among animals with nervous systems in that they lack the canonical RIM, bearing only the newly identified homolog. Through phylogenetic analysis, we find that CaV2 channel D/E-D/E/H-WC-COOH like PDZ ligand motifs were present in the common ancestor of cnidarians and bilaterians, and delineate some deeply conserved C-terminal structures that distinguish CaV1 from CaV2 channels, and CaV1/CaV2 from CaV3 channels.

A lethal fungal pathogen directly alters tight junction proteins in the skin of a susceptible amphibian.

Bacterial and viral pathogens can weaken epithelial barriers by targeting and disrupting tight junction (TJ) proteins. However, comparatively little is known about the direct effects of fungal pathogens on TJ proteins and their expression. The disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is threatening amphibian populations worldwide. Bd is known to infect amphibian skin and disrupt cutaneous osmoregulation. However, exactly how this occurs is poorly understood. This study considered the impact of Bd infection on the barrier properties of the Australian green tree frog (Litoria caerulea) epidermis by examining how inoculation of animals with Bd influenced the paracellular movement of FITC-dextran (4 kDa, FD-4) across the skin in association with alterations in the mRNA and protein abundance of select TJ proteins of the epidermal TJ complex. It was observed that Bd infection increased paracellular movement of FD-4 across the skin linearly with fungal infection load. In addition, Bd infection increased transcript abundance of the tricellular TJ (tTJ) protein tricellulin (Tric) as well as the bicellular TJ (bTJ) proteins occludin (Ocln), claudin (Cldn)-1, Cldn-4 and the scaffolding TJ protein zonula occludens 1 (ZO-1). However, while Tric protein abundance increased in accord with changes in transcript abundance, protein abundance of Cldn-1 was significantly reduced and Ocln protein abundance was unchanged. Data indicate that disruption of cutaneous osmoregulation in L. caerulea following Bd infection occurs, at least in part, by an increase in epidermal paracellular permeability in association with compromised integrity of the epidermal TJ complex.

Claudin tight junction proteins in rainbow trout (Oncorhynchus mykiss) skin: Spatial response to elevated cortisol levels.

This study examined regional distribution and corticosteroid-induced alterations of claudin (cldn) transcript abundance in teleost fish skin. Regional comparison of mRNA encoding 20 Cldns indicated that 12 exhibit differences in abundance along the dorsoventral axis of skin. However, relative abundance of cldns (i.e. most to least abundant) remained similar in different skin regions. Several cldns appear to be present in the epidermis and dermal vasculature whereas others are present only in the epidermis. Increased circulating cortisol levels significantly altered mRNA abundance of 10 cldns in a region specific manner, as well as corticosteroid receptors and 11ß-hydroxysteroid dehydrogenase (type 2). Epidermis and epidermal mucous cell morphometrics also altered in response to cortisol, exhibiting changes that appear to enhance skin barrier properties. Taken together, data provide a first look at spatial variation in the molecular physiology of the teleost fish integument TJ complex and region-specific sensitivity to an endocrine factor.