Eukaryote voltage-gated Ca²⁺ channels of the Ca_V2 channel family are hetero-oligomers formed by the pore-forming Ca_Vα1 protein assembled with auxiliary Ca_Vα2δ and Ca_Vβ subunits. Ca_Vβ 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_Vα1 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_Vβ. Recombinant expression of its cDNA with mammalian high-voltage activated Ca²⁺ channel Ca_V2.3 in mammalian HEK cells produced robust voltage-gated inward Ca²⁺ currents with typical activation and inactivation properties. Like Ca_Vβ, Sroβ prevents fast degradation of total Ca_V2.3 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_Vα1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of Ca_V2.3 with Sroβ and functional upregulation of Ca_V2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroβ, similar to the locus found in mammalian Ca_Vβ, 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_Vβ through a minimally conserved molecular interface.

真核生物电压门控的Ca ²⁺的钙通道_V 2通道家族是由孔形成的Ca形成异寡聚体_V与辅助的Ca组装α1蛋白_V α2δ和Ca _V β亚基。的Ca _V β亚基由Src同源3（SH3）结构域，并通过HOOK域连接的鸟苷酸激酶（GK）结构域形成。该GK结构域结合的孔形成的Ca的保守胞质区_V α1亚单位中被称为“AID”​​。在这里，我们通过研究从鞭毛鞭毛虫克隆的MAGUK蛋白（XM_004990081）的功能，探索了远距离真核生物中Ca _V通道亚基之间的系统发生和功能关系。Salpingoeca rosetta（Sro）。此MAGUK蛋白（Sroβ）设有SH3和GK用25％一级序列同一性的哺乳动物的Ca的结构域_V β。在哺乳动物HEK细胞中，其cDNA与哺乳动物高压激活的Ca ²⁺通道Ca _V 2.3的重组表达产生了具有典型激活和失活特性的强大的电压门控内向Ca ²⁺电流。如Ca _V β，Sroβ防止总的Ca的快速降解_V在放线菌酮测定法2.3的蛋白质。三维同源模型预测Sroβ的GK结构域与成孔Ca _V的AID基序之间的相互作用α1蛋白。替代AID残基Trp（W386A）和Tyr（Y383A）会严重损害Ca _V 2.3与Sroβ的共免疫沉淀以及Ca _V 2.3电流的功能上调。同样地，Sroβ的GK域内的6个残基的缺失，类似于轨迹在哺乳动物中发现的Ca _V β，显著降低的峰值电流密度。总之我们的数据表明，一个祖先MAGUK蛋白重新构成负责信道上调哺乳动物钙生物物理和分子特性_V通过最低限度地保守的分子界面β。