Identification of novel functional sites in the Ca _v 1.3 calcium channel α1-subunit using evolutionary modeling

Voltage-gated calcium channels (VGCCs) regulate differentiation, function, and survival of excitable cells, and pathogenic variants cause diverse disorders. Most known disease-associated VGCC mutations affect well-characterized regions controlling voltage-dependent gating and channel kinetics, while many residues remain functionally unannotated. We developed an evolutionary model to predict the pathogenic potential of residues in the pore-forming Ca v 1.3 subunit, previously validated with de novo gain-of-function variants linked to neurodevelopmental diseases. Here we show that the model recapitulates established functional regions and prospectively identifies functional sites at single–amino acid resolution. Electrophysiological analyses of five predicted variants across multiple channel domains confirmed functional alterations. The approach also captures loss-of-function variants typically pathogenic only in the homozygous state, establishing a predictive framework for identifying and functionally characterizing pathogenic variants in Ca v 1.3 and related ion channels.