DIV W[+2]A mutation increases local conformational flexibility near the selectivity filter. (A) MD simulations were used to estimate the local conformational flexibility of the DIV pore helices of wild-type Ca_V1.3 embedded in a lipid bilayer. Colormap indicates computed B-factors for residues in DIV pore helices ranging from 0 (green) to 147.7 (red) corresponding to increasing flexibility. (B) DIV W[+2]A mutation increases local conformational flexibility. (C and D) In comparison to wild-type channels (panel C), simulations of DI W[+2]A mutant (panel D) show minimal change in computed B-factors for residues in DII pore helices. (E and F) Both wild-type (panel E) and DII W[+2]A mutant (panel F) show minimal change in local conformational flexibility. (G and H) Similarly, both wild-type (panel G) and DII W[+2]A mutant (panel H) exhibit minimal change in local conformational flexibility. This differential effect between DI–III versus DIV points to a specific role for DIV in tuning VDI. (I) Left: Comparison of wild-type versus DIV W[+2]A mutation reveals key changes in the selectivity filter conformation. The DIV W[+2]A mutation alters the arrangement of Ca²⁺ coordinating E[0] residue, with DIV E[0] residue shifted by ∼4 Å. Right: Detailed view of the DIV E[0] residue (E1402).