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 Ca(v)2 channel family are hetero-oligomers formed by the pore-forming Ca-v alpha 1 protein assembled with auxiliary Ca-v alpha 2 delta and Ca-v beta subunits. Ca-v beta 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 Ca-v alpha 1 subunit referred as the "AID". Herein we explored the phylogenetic and functional relationship between Ca-v 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 (Sro beta) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian Ca-v beta. Recombinant expression of its cDNA with mammalian high-voltage activated Ca2+ channel Ca(v)2.3 in mammalian HEK cells produced robust voltage-gated inward Ca2+ currents with typical activation and inactivation properties. Like Ca-v beta, Sro beta prevents fast degradation of total Ca(v)2.3 proteins in cycloheximide assays. The three-dimensional homology model predicts an interaction between the GK domain of Sro beta and the AID motif of the pore-forming Ca-v alpha 1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of Ca(v)2.3 with Sro beta and functional upregulation of Ca(v)2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sro beta, similar to the locus found in mammalian Ca-v beta, 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 Ca-v beta through a minimally conserved molecular interface.