Asymmetric role of D IV pore helix in tuning VDI of Ca _V 1.3. (A) Three distinct end-stage mechanisms for Ca_V1.3 inactivation. Scheme 1 shows a hinged-lid mechanism where the pore is occluded by an inactivation particle (DI–DII linker), scheme 2 shows an allosteric mechanism where the open probability is reduced through increased channel closure, and scheme 3 shows selectivity filter collapse. (B) Sequence alignment shows the conservation of a tryptophan residue (W) in the pore segment adjacent to the putative Ca²⁺ coordinating glutamate (E) residue. (C) Structural models show the pseudo-tetrameric architecture of Ca_V1.3 (PDB ID 7UHG). The inset shows the ion conduction pathway lined by the selectivity filter Ca²⁺ coordinating glutamate residues and the spatial location of the conserved tryptophan. (D) Representative Ba²⁺ current traces evoked in response to an 800-ms voltage step to 0 mV show minimal VDI of both wild-type (gray) and DI W[+2]A mutant channels. The wild-type current is normalized to compare inactivation kinetics. (E and F) Similarly, both DII W[+2]A and DIII W[+2]A mutant channels also showed minimal VDI. Format as in panel C. (G) By contrast, DIV W[+2]A mutation enhanced VDI of Ca_V1.3. Format as in panel C. (H) Population data shows the fraction of remaining current following 800-ms depolarization (r₈₀₀). The DIV W[+2]A mutation selectively increases inactivation in comparison to both wild-type (gray) and W[+2]A substitutions in DI–DIII. Each bar, mean ± SEM. Wild-type, n = 5 cells, DI W[+2]A, n = 5 cells, DII W[+2]A, n = 6 cells, DIII W[+2]A, n = 5 cells, DIV W[+2]A, n = 7 cells. ***P < 0.001 by Tukey’s multiple comparisons test.