To understand how splicing factors regulate alternative splicing (AS) of the voltage-gated calcium channel (VGCC) CaV1.3 and whether AS modifies its electrophysiological properties.

The VGCC CaV1.3 subunit, encoded by CACNA1D, is highly expressed in neurons, endocrine cells, and cardiac tissue. Mutations in CACNA1D are associated with primary aldosteronism with seizures and neurologic abnormalities (PASNA), bradycardia, and autism spectrum disorder. Additionally, CaV1.3-mediated calcium influx exacerbates motor neuron hyperexcitability following spinal cord injury (SCI). Alternative splicing at exons 44 and 45 generates a full-length “Long” (44-45L) isoform and a “Delta” (44-45Δ) isoform lacking both exons. These isoforms differ in gating and drug sensitivity, yet the electrophysiological and pharmacological properties of many CaV1.3 splice variants remain largely unexplored.

Mouse Cacna1d minigene reporters were engineered to assess the regulation of alternatively spliced exons. Electrophysiological properties of CaV1.3 variants were analyzed by patch clamp in mammalian cells and two-electrode voltage clamp in Xenopus oocytes.

Splicing factors, including Ptbp2, Mbnl2, Rbfox1, and Rbfox2, modulated distinct CaV1.3 splicing events. The Delta isoform displayed reduced current amplitudes compared to the Long isoform in both Ba²⁺ and Ca²⁺ bath solutions. Quantitative analyses revealed a significant reduction in peak current density (I_max) in the Delta isoform under both ionic conditions.

Our research suggests that alternative splicing markedly alters CaV1.3 channel function, reshaping its biophysical properties and potentially influencing its pathological role in neurological disease. By linking splicing regulation with channel physiology, this work provides novel mechanistic insights and potential therapeutic entry points for CaV1.3-related disorders.