Molecular Dynamics Simulations of Ion Permeation in Human Voltage-Gated Sodium Channels

Abstract

The recent determination of cryo-EM structures of voltage-gated sodium (Na_v) channels has revealed many details of these proteins. However, knowledge of ionic permeation through the Na_v pore remains limited. In this work, we performed atomistic molecular dynamics (MD) simulations to study the structural features of various neuronal Na_v channels based on homology modeling of the cryo-EM structure of the human Na_v1.4 channel and, in addition, on the recently resolved configuration for Na_v1.2. In particular, single Na⁺ permeation events during standard MD runs suggest that the ion resides in the inner part of the Na_v selectivity filter (SF). On-the-fly free energy parametrization (OTFP) temperature-accelerated molecular dynamics (TAMD) was also used to calculate two-dimensional free energy surfaces (FESs) related to single/double Na⁺ translocation through the SF of the homology-based Na_v1.2 model and the cryo-EM Na_v1.2 structure, with different realizations of the DEKA filter domain. These additional simulations revealed distinct mechanisms for single and double Na⁺ permeation through the wild-type SF, which has a charged lysine in the DEKA ring. Moreover, the configurations of the ions in the SF corresponding to the metastable states of the FESs are specific for each SF motif. Overall, the description of these mechanisms gives us new insights into ion conduction in human Na_v cryo-EM-based and cryo-EM configurations that could advance understanding of these systems and how they differ from potassium and bacterial Na_v channels.

Figure 1

(A) Equilibrated homology-based Na_v1.2 model. The domains are colored as in refs (−65): Domain I, gray; Domain II, yellow; Domain III, green; Domain IV, cyan. This color scheme is applied throughout the article. (B) Extracellular view of the model. (C) Representation of the asymmetric Na_v1.2 SF. The SF vestibule is enclosed by the side chains of the D_IE_IIK_IIIA_IV motif (D384, E942, K1422, A1714) and the internal carbonyl oxygen atoms of the two preceding residues in each repeat. The residues of the external E_IE_IID_IIID_IV motif (E387, E945, D1426, D1717), above the DEKA domain, are also shown. The EEDD domain and the SF region form the C/SF.

Figure 2

Superposition between the pore of the template (PDB ID 6AGF, hNa_v1.4, pink ribbons) and the equilibrated homology-based Na_v1.2 model. The domains are colored according to the convention introduced in Figure 1. The side chains belonging to the C/SF are shown as sticks. The residues are labeled following the Na_v1.2 enumeration.

Figure 3

Sodium ion resolved in the SF of the PDB ID 6J8E Na_v1.2 cryo-EM structure. The pore blocker μ-conotoxin KIIIA is not shown.

Figure 4

(A) Equilibrated homology-based Na_v1.2 α subunit embedded in a hydrated POPC bilayer (brown wires), surrounded by water (red and white). Sodium (yellow) and chloride (blue) ions are represented as spheres. (B) Equilibrated homology-based Na_v1.2 pore-only domain embedded in the same environment as described in (A).

Figure 5

Main simulations performed in this work. For each MD run, the integration time step Δt is shown. The meaning of OTFP₁ and OTFP₂ is specified in the text. Results of additional simulations are included in the Supporting Information. The list of all the simulations included in this work is reported in Tables S5 and S6.

Figure 6

Histograms of the z coordinate values of (A) a single Na⁺ ion in the C/SF, (B) the ammonium nitrogen of the DEKA ring, and (C) two Na⁺ ions and their COM in the double occupancy events. Vertical lines indicate the z coordinates of the Cα atoms of C/SF residues from the equilibrated configuration of Na_v1.2 (red for EEDD, black for DEKA, and blue for each couple of residues right below the DEKA ring).

Figure 7

FESs for single Na⁺ translocation for each of the four systems, as indicated in the labels (DEK⁺¹A, DEK⁰A, DEE^–1A, DEE⁰A). Yellow dots indicate the sets of snapshots used for the representative configurations y₁ to y₇. For each set y_i, the backbone of the various repeats is shown in ribbons, using the color code illustrated in Figure 1. For clarity, a fragment of Domain IV (cyan) is not drawn. The transparent surfaces represent the volume occupancy of Na⁺ and the tip atoms of the DEKA and EEDD side chains, and they are colored according to atomic charge (red for negative charges and blue for positive charges). The surface of residue 1422 is colored cyan for DEK⁰A and orange for DEE⁰A).

Figure 8

(A) MFEP (white dots) of the DEK⁺¹A system’s averaged FES, superimposed on the corresponding map. (B) FE profile along the MFEP.

Figure 9

FESs for translocation of two Na⁺ ions in the SF of each of the four systems, as indicated in the labels (DEK⁺¹A, DEK⁰A, DEE^–1A, DEE⁰A). Cyan dots indicate the snapshots used for the representative configurations z₁ to z₆, which are reported as explained in Figure 7.