Fig 2: 3D model of the mutant Cav3.2 channel reveals significant structural changes.A Predicted topologies of the wild type (WT) and mutant (MUT) Cav3.2 channels showing the four homologous domains (I–IV) in different colors, each composed of six transmembrane segments (bars S1–S6). The 52-amino acid insertion (yellow) is located between segments S5 and S6 of domain II (between residues Q969 and I970). The hydropathy profiles of the amino acid sequences are shown below the channel topologies. B Structural features of the WT and MUT Cav3.2 channels. Bi Simulations of the proteins viewed from the cytosolic side of the plasma membrane are shown in cartoon representation and colored by domain (DI–DIV). Domain II is colored in magenta and the 52-amino acid insertion is detached in yellow. Bii Structural view of the pore center showing the differences in the pore diameter between WT and MUT Cav3.2 channels.

Fig 3: Mutant Cav3.2-mediated hyperfunction of mTORC1 signaling acts alone to cause enhanced proliferation of F2688-1-derived NPCs and together with impaired Reelin signaling in the abnormal migration of these cells.A Representative phase-contrast microscopy photographs of control- and F2688-1-derived NPCs. The bar graph shows the median cell body size (including soma size and all cell surface projections) of control-derived NPCs (n = 4) and F2688-1-derived NPCs (NPCs derived from 2 iPSC clones). There were no significant differences in median body size between the groups. B Line graph showing cell proliferation curves (live cell numbers at Day 0, 48 h, 72 h) of control-derived NPCs (n = 4) and F2688-1-derived (NPCs derived from 2 iPSC clones) cultured in the presence of either vehicle, 1 µM of NNC 55-0396 (NNC), or 100 nM of rapamycin; and in the presence of either mock-conditioned medium (mock) or Reelin-conditioned medium (Reelin). The graph shows the median value and interquartile range for each group. F2688-1 NPCs show significantly higher proliferation rates compared with control NPCs, which were completely rescued by treatment with either rapamycin or NNC. On the other hand, treatment with Reelin had no effect on the proliferation of F2688-1 NPCs. C Line graph showing the percentage relative wound density (RWD) over time in control-derived NPCs (n = 4) and in F2688-1-derived NPCs (NPCs derived from 2 iPSC clones) cultured in the presence of either vehicle, 1 µM of NNC, or 100 nM of rapamycin; and in the presence of either mock or Reelin. The graph shows the median value and interquartile range for each group. F2688-1 NPCs exhibit significantly higher migration rates compared with control NPCs, which was significantly attenuated by treatment with rapamycin, and completely rescued to levels equal to those of the untreated control NPCs in the exponential phase of the healing curve by treatment with either NNC or Reelin (analyzed time interval: 15 h–25 h). **p = 0.01, ****p = 0.0001.

Fig 4: Splice donor site variant in intron 13 of the CACNA1H gene in patient F2688-1 results in retention of the entire intron in the mature mRNA.A Schematic representation of intron 13 (In13) retention in the CACNA1H transcript showing the locations of primers for conventional RT-PCR. The PCR band corresponding to the wild-type allele is 370 bp long, whereas the band corresponding to the mutant allele is 526 pb long due to retention of intron 13 in the mature transcript. B Representative image of agarose gel electrophoresis showing conventional RT-PCR products. The 526 bp PCR product of the mutant allele is only observed in F2688-1-derived NPCs (n = 2 iPSC clones-derived NPCs). C Sanger sequencing of the cloned 526 bp PCR band confirmed intron 13 retention in the mature CACNA1H transcript. The location of the variant NM_001005407:c.2907+1 G>A is shown. D Quantitative RT-PCR demonstrated that the relative transcription levels of CACNA1H are not changed in F2688-1-derived NPCs (n = 2 iPSC clones-derived NPCs) compared with control NPCs (Controls, n = 4). The bar graph shows the median value and interquartile range for each group.

Fig 5: HEK293T cells overexpressing mutant Cav3.2 channel show increased Ca2+ influx, hyperfunctional mTORC1 signaling, and impaired activation of a Reelin signaling component.HEK293T cells were transfected with an empty vector (EMPTY-vector; n = 5) or with plasmids expressing either wild-type (WT-a1Ha; n = 5) or mutant (MUT-a1Ha; n = 5) Cav3.2 channels. A Intracellular Ca2+ measurements in transfected cells depolarized with 100 mM KCl and cultured in the absence (vehicle) or presence of 10 µM of the T-type Ca2+ channels blocker NNC 55-0396 (NNC). The bar graph shows the median value and interquartile range for each group. Cells overexpressing mutant Cav3.2 show significantly increased Ca2+ influx, which was rescued by treatment with NNC. Only the p-values obtained from the analyses using MUT-a1Ha are shown. B Representative immunoblots showing the expression levels of pRPS6, pSRC, SRC, and ß-actin (used as loading control) in transfected cells cultured in the absence (-) or presence (+) of 10 µM of NNC. The bar graph shows the median levels and interquartile ranges of normalized pRPS6 and pSRC for each group; only the p-values obtained from the analyses using MUT-a1Ha are shown. Cells overexpressing mutant Cav3.2 show significantly increased pRPS6 levels and significantly decreased pSRC levels, which was rescued by treatment with NNC. *p < 0.05, **p = 0.01, ****p = 0.0001.