An ancestral MAGUK protein supports the modulation of mammalian voltage-gated Ca2+ channels through a conserved CaVβ–like interface

Eukaryote voltage-gated Ca2+ channels of the CaV2 channel family are hetero-oligomers formed by the pore-forming CaVα1 protein assembled with auxiliary CaVα2δ and CaVβ subunits. CaVβ subunits are formed by a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain connected through a HOOK domain. The GK domain binds a conserved cytoplasmic region of the pore-forming CaVα1 subunit referred as the “AID”. Herein we explored the phylogenetic and functional relationship between CaV channel subunits in distant eukaryotic organisms by investigating the function of a MAGUK protein (XM_004990081) cloned from the choanoflagellate Salpingoeca rosetta (Sro). This MAGUK protein (Sroβ) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian CaVβ. Recombinant expression of its cDNA with mammalian high-voltage activated Ca2+ channel CaV2.3 in mammalian HEK cells produced robust voltage-gated inward Ca2+ currents with typical activation and inactivation properties. Like CaVβ, Sroβ prevents fast degradation of total CaV2.3 proteins in cycloheximide assays. The three-dimensional homology model predicts an interaction between the GK domain of Sroβ and the AID motif of the pore-forming CaVα1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of CaV2.3 with Sroβ and functional upregulation of CaV2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroβ, similar to the locus found in mammalian CaVβ, significantly reduced peak current density. Altogether our data demonstrate that an ancestor MAGUK protein reconstitutes the biophysical and molecular features responsible for channel upregulation by mammalian CaVβ through a minimally conserved molecular interface. [Display omitted] •Little is known about voltage-gated ion channels in unicellular choanoflagellates.•MAGUK protein was cloned from Salpingoeca rosetta.•The 381-residue protein upregulates mammalian calcium channels.•The mechanism of interaction emerged before the animal-sponge split.•This shed a novel perspective on the evolution of ion channels.