Identification of novel functional sites in the Ca_v1.3 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_v1.3 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_v1.3 and related ion channels.