Changes in Ion Selectivity Following the Asymmetrical Addition of Charge to the Selectivity Filter of Bacterial Sodium Channels

Abstract

Voltage-gated sodium channels (NaVs) play fundamental roles in eukaryotes, but their exceptional size hinders their structural resolution. Bacterial NaVs are simplified homologues of their eukaryotic counterparts, but their use as models of eukaryotic Na⁺ channels is limited by their homotetrameric structure at odds with the asymmetric Selectivity Filter (SF) of eukaryotic NaVs. This work aims at mimicking the SF of eukaryotic NaVs by engineering radial asymmetry into the SF of bacterial channels. This goal was pursued with two approaches: the co-expression of different monomers of the NaChBac bacterial channel to induce the random assembly of heterotetramers, and the concatenation of four bacterial monomers to form a concatemer that can be targeted by site-specific mutagenesis. Patch-clamp measurements and Molecular Dynamics simulations showed that an additional gating charge in the SF leads to a significant increase in Na⁺ and a modest increase in the Ca²⁺ conductance in the NavMs concatemer in agreement with the behavior of the population of random heterotetramers with the highest proportion of channels with charge -5e. We thus showed that charge, despite being important, is not the only determinant of conduction and selectivity, and we created new tools extending the use of bacterial channels as models of eukaryotic counterparts.

Keywords: computer simulations; ion channel; patch-clamp; permeability; selectivity.

Figure 1

Na⁺/Ca²⁺ selectivity for NaChBac monomer mixtures. The voltage–current relations (A,B) and the mean (+/− SEM) whole-cell peak current density at −10 mV (C,D) for Na⁺ (A,C) and Ca²⁺ (B,D) in CHO cells transfected with cDNAs encoding for NaChBac channels possessing either a wild-type selectivity filter (LESWAS/LES) or a mutated selectivity filter (LASWAS/LAS or LEDWAS/LED); 5 µg of total DNA was used per transfection and was composed of either a mixture of types of cDNA at defined ratios, as indicated in Table 1 and on the X-axis, or a single cDNA type. Numbers in parentheses indicate the number of replicates.

Figure 2

Na⁺/Ca²⁺ selectivity for NavMs concatemer possessing varied Q_f values in their SF. The original recordings representatives of wild-type NavMS (A) and its DI (B) and DII (C) mutants in 140 mM Na⁺ solution (grey traces) and in 100 mM Ca²⁺ solution (black traces). The voltage–current relations (D,E) and the mean (+/− SEM) whole-cell peak current density at −10 mV (F,G) for Na⁺ (D,F) and Ca²⁺ (E,G) in HEK 293T cells transfected with cDNAs encoding for either wild-type or mutated NavMS. Numbers in parentheses indicate the number of replicates; and in HEK293T cells transfected with wild-type NavMS concatemer (WT) and mutant NavMS concatemer (DI and DII).

Figure 3

MD simulations for the WT and mutant NavMS in NaCl 140 mM. (a,b) Potential of Mean Force of Na⁺ as a function of the axial position in WT NavMS (a) and the mutant with charge Q_f = −5e (b). (c,d) Average number of coordinating oxygens per sodium ion in axial bins with a thickness of 2.0 Å. (c) Wild-type NavMs; (d) NavMs mutant with Q_f = −5e. The distance cutoff to identify sodium-chloride interactions was set to 3.5 Å, and for sodium-oxygen to 3.2 Å. Color code is as follows. Blue line: number of coordinating water-provided oxygens; green line: number of coordinating oxygens provided by aspartate and glutamates; red line; number of coordinating oxygens provided by other protein residues; black line: total number of coordinating oxygens; magenta line: number of coordinating chlorides. (e,h) Configuration of the selectivity filter of wild-type NavMS (e,g) and the mutant with charge Q_f = −5e (f,h). All the structures correspond to the last frame of a 100 ns simulation in 0.14 M NaCl. Panels (e,f) show a side view of the SF; panels (g,h) show the top view. Glu178 is shown in red, while Asp179 is shown in purple. The backbone of Leu177 is shown in green. Sodium ions are portrayed as blue beads. Panels (e,g) also show the water molecules that mediate the interactions between the resident sodium ion and the protein in wild-type NavMS.

Figure 4

MD simulations for WT and mutant NavMs in CaCl₂ 100 mM. (a,b) Potential of Mean force of Ca²⁺ as a function of the axial position in WT NavMS (a) and the mutant with charge Q_f = −5e (b). (c,d) Average number of coordinating oxygens per calcium ion in axial bins with a thickness of 2.0 Å. (c) Wild-type NavMs; (d) NavMs mutant with Q_f = −5e. The distance cutoff to identify both calcium-chloride and calcium-oxygen interactions was set to 3.5 Å. Color code is as follows. Blue line: number of coordinating water oxygens; green line: number of coordinating oxygens provided by aspartate and glutamates; red line; number of coordinating oxygens provided by other protein residues; black line: total number of coordinating oxygens; magenta line: number of coordinating chlorides. (e–h) Configuration of the selectivity filter of wild-type NavMS (e,g) and the mutant with charge Q_f = −5e (f,h). All the structures correspond to the last frame of a 150 ns simulation in 0.10 M of CaCl₂. Panels (e,f) show a side view of the SF; panels (g,h) show the top view. Glu178 is shown in red, while Asp179 is shown in purple. The backbone of Leu177 is shown in green. Calcium ions are portrayed as orange beads.