Early metazoan origin and multiple losses of a novel clade of RIM pre-synaptic calcium channel scaffolding protein homologues

Abstract

AbstractThe precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which,Rab3 interactingmolecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channelsin vitrovia 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 placozoanTrichoplax adhaerens, and molluscs. RNA expression and localization studies inTrichoplaxand the mollusc snailLymnaea stagnalisindicate differential regional/tissue type expression, but overlapping expression in single isolated neurons fromLymnaea. 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-COOHlike 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.