An ancestral MAGUK protein supports the modulation of mammalian voltage-gated Ca 2+ channels through a conserved Ca V β-like interface.

Eukaryote voltage-gated Ca ²⁺ channels of the Ca _V 2 channel family are hetero-oligomers formed by the pore-forming Ca _V α1 protein assembled with auxiliary Ca _V α2δ and Ca _V β subunits. Ca _V β 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 α1 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 (Sroβ) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian Ca _V β. Recombinant expression of its cDNA with mammalian high-voltage activated Ca ²⁺ channel Ca _V 2.3 in mammalian HEK cells produced robust voltage-gated inward Ca ²⁺ currents with typical activation and inactivation properties. Like Ca _V β, Sroβ 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 Sroβ and the AID motif of the pore-forming Ca _V α1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of Ca _V 2.3 with Sroβ and functional upregulation of Ca _V 2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroβ, similar to the locus found in mammalian Ca _V β, 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 β through a minimally conserved molecular interface.
Competing Interests: Declaration of competing interest The authors declare that they do not have any conflict of interest with the contents of this manuscript.
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