Gating defects of disease-causing de novo mutations in Ca(v)1.3 Ca(2+) channels

Recently, we and others identified somatic and germline de novo gain-of-function mutations in CACNA1D, the gene encoding the α1-subunit of voltage-gated Ca(v)1.3 Ca(2+)-channels. While somatic mutations identified in aldosterone producing adenomas (APAs) underlie treatment-resistant hypertension, germline CACNA1D mutations are associated with a neurodevelopmental disorder characterized by a wide symptomatic spectrum, including autism spectrum disorder. The number of newly identified CACNA1D missense mutations is constantly growing, but their pathogenic potential is difficult to predict in silico, making functional studies indispensable to assess their contribution to disease risk. Here we report the functional characterization of previously identified CACNA1D APA mutations F747L and M1354I using whole-cell patch-clamp electrophysiology upon recombinant expression in tsA-201 cells. We also investigated if alternative splicing of Ca(v)1.3 affects the aberrant gating of the previously characterized APA mutation R990H and two mutations associated with autism spectrum disorder (A479G and G407R). Splice-variant dependent gating changes are of particular interest for germline mutations, since the relative expression of Ca(v)1.3 splice variants differs across different tissues and within brain regions and might therefore result in tissue-specific phenotypes. Our data revealed a complex gain-of-function phenotype for APA mutation F747L confirming its pathogenic role. Furthermore, we found splice-variant dependent gating changes in R990H, A749G and G407R. M1354I did not change channel function of Ca(v)1.3 splice variants and should therefore be considered a rare non-pathogenic variant until further proof for its pathogenicity is obtained. Our new findings together with previously published data allow classification of pathogenic CACNA1D mutations into four categories based on prototypical functional changes.