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

Abstract

The precise localization of Ca V 2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which, Rab3 interacting molecule or RIM is particularly important. In vertebrates, RIM has been shown to interact with Ca V 2 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 . Here, we describe a previously undescribed 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 Ca V 2 channel D/E-D/E/H-WC-COOH like PDZ ligand motifs were present the common ancestor of cnidarians and bilaterians. We also delineate some deeply conserved C-terminal structures that distinguish Ca V 1 from Ca V 2 channels, and Ca V 1/Ca V 2 from Ca V 3 channels.Average counts of fluorescent granules within regions of the animal characterized by distinct celltype content (i.e. the edge, fiber cell zone and lipophil cell zone (Mayorova, et al. 2019)), revealed that II-RIM is more abundantly expressed than I-RIM overall and in each region ( Figure 1F; p-values for Tukey's tests after one-way ANOVAs: edge <0.05; fiber zone <0.0005; lipophil zone <0.00005; sum of regions <0.00005; ANOVA for separate regions: df = 5, F = 17.1, p = 2.1E-15; ANOVA for regions combined: df = 1, F = 24.5, p = 2.1E-6). Notably, these patterns are consistent with mRNA expression levels measured as average transcripts per million (TPM) in the transcriptome data, where II-RIM is more abundantly expressed than I-RIM at the whole animal level ( Figure 1B). Also consistent were the average TPM values and counted granules for in situ hybridization of the three Trichoplax Ca V channels (Ca V 1-Ca V 3), where TPM and granule counts for Ca V 1 were significantly higher compared to Ca V 2 and Ca V 3 within edge and lipophil zones and all three regions combined (i.e. compare Figure 1B and F) (p-values for Tukey's tests after one-way ANOVAs of granule counts: Ca V 1 vs. Ca V 2 edge <0.00005; Ca V 1 vs.Ca V 2 lipophil zone <0.00005; Ca V 1 vs. Ca V 2 total <0.00005; Ca V 1 vs. Ca V 3 edge <0.00005; Ca V 1 vs.Ca V 3 lipophil zone <0.00005; Ca V 1 vs. Ca V 3 total <0.00005; ANOVA for separate regions: df = 8, F = 86.8, p = 0; ANOVA for regions combined: df = 2, F = 159.0, p = 0;). Indeed, in spite of...