Mint/X11 PDZ domains from non-bilaterian animals recognize and bind Ca _V 2 calcium channel C-termini in vitro

PDZ domain mediated interactions with voltage-gated calcium (Ca _V ) channel C-termini play important roles in localizing membrane Ca ²⁺ signaling. The first such interaction was described between the scaffolding protein Mint-1 and Ca _V 2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a 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. In addition to Ca _V 2 channels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast 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 in dissociated neurons from the cnidarian Nematostella vectensis . Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly binds 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 suggest that the capacity of Mint to bind CaV2 channels predates pre-bilaterian animals, and that evolutionary changes in Ca _V 2 channel C-terminal sequences resulted in altered binding modalities with Mint.