Fig. 5: The EEKE selectivity filter of NALCN.

a Overall structure of the pore domain of NALCN. Side views of the pore domain from the diagonal repeats are shown. The EEKE residues in the selectivity filter (SF) are shown in stick. Repeat I and repeat III adopt large extracellular loops, reminiscent of Ca_v1.1. A π helix appears in each S6 segment and is indicated by red arrows. b Sequence alignment of the SF and the connecting pore helices among NALCN, Ca_v1.1, and Na_v1.4. The critical EEKE (E280, E554, K1115, and E1389) residues in NALCN and the corresponding residues in Ca_v1.1 (EEEE) and Na_v1.4 (DEKA) are shaded yellow. The invariant Trp residues and the highly conserved Thr residues are colored light green. The acidic residues that may help to coordinate cations in NALCN are shaded orange. c Structure comparisons of the SF between NALCN and Na_v1.4 (left) or Ca_v1.1 (right). The critical residues in the SF are shown in the stick. d Top view and two side views of sodium probability density generated from pore domain equilibration. Three potential Na⁺ binding sites were identified and labeled in purple. The most stable binding site (Site1) is signposted by a Na⁺ ion (magenta sphere). Please also refer to Supplementary Movie for the illustrative trajectory of equilibration. e Ion numbers within 4 Å of SF residues (EEKE) over time (left) and the probability statistics (right). The statistics represent the results of three independent simulations. f Structural comparison of Site 3 (EED motif) in NALCN and the Na⁺ binding site (DEE motif) in Na_v1.4 (top) and of Site1 in NALCN and a Ca²⁺ binding site in Ca_v1.1 (bottom). Na⁺ and Ca²⁺ are shown as purple and green spheres, respectively.