Mechanistic insights for VDI. (A) VDI perturbations mapped onto the Ca_V1.3 homology model from Fig. 1 B. Residues outside of mutagenesis screen shown in gray. Colored regions indicate VDI₃₀₀ upon mutation to proline. See color legend to right, and note that native channel VDI₃₀₀ is ∼0.1 (blue). (B and C) Proposed mechanism of VDI in Ca_V1.3. Native channels (B) have very little VDI, likely due to the presence of a shield (red), which prevents the hinged lid (blue) from reaching its binding site (green). VDI-enhancing S6 mutations likely disrupt this shield (C). (D and E) Sensitivity of VDI to β subunits supports the notion of a shield in Ca_V1.3. Although β_1b typically enhances VDI by allowing greater hinged-lid mobility than β_2a (cartoon in D), native Ca_V1.3 and non-hotspot mutant N1462P have no such effect (D). In contrast, if the putative shield is perturbed (two mutants in E), β_1b significantly enhances VDI.