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 homologous domain architectures as known RIM homologues, but 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 homologue. 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.

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.

No abstract

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.