Structural basis of human Nav1.5 gating mechanisms

Abstract

Voltage-gated Na_v1.5 channels are central to the generation and propagation of cardiac action potentials. Aberrations in their function are associated with a wide spectrum of cardiac diseases including arrhythmias and heart failure. Despite decades of progress in Na_v1.5 biology, the lack of structural insights into intracellular regions has hampered our understanding of its gating mechanisms. Here, we present two cryo-EM structures of human Na_v1.5 in open states, revealing sequential conformational changes in gating charges of the voltage-sensing domains (VSDs) and several intracellular regions. Despite the channel being in the open state, these structures show repositioning, but no dislodging of the IFM motif in the receptor site. Molecular dynamics analyses show our structures with CTD conduct Na⁺ ions. Notably, our structural findings highlight a dynamic C-terminal domain (CTD) and III-IV linker interaction, which regulates the conformation of VSDs and pore opening. Electrophysiological studies confirm that disrupting this interaction alters fast inactivation of Na_v1.5. Together, our structure-function studies establish a foundation for understanding the gating mechanisms of Na_v1.5 and the mechanisms underlying CTD-related channelopathies.

Keywords: action potentials; arrhythmias; cryo-EM; electrophysiological studies; sodium channels.

Fig. 1.

Cryo-EM structures of full-length hNa_v1.5. (A) Side view of the cryo-EM reconstruction of Model-I. Individual domains and interdomain linkers are segmented and color-coded. The Lower panel depicts the atomic structure of Model-I including the resolved CTD, interdomain linkers, lipid molecules, and covalently attached glycans. The structural features are segmented and color-coded according to the density map. (B) Side view of the cryo-EM reconstruction (Top) and atomic structure (Bottom) of Model-II. Color-coded according to (A).

Fig. 2.

Insights into key functional regions of hNa_v1.5. (A) The intracellular view of the structural superimposition of Model-II (bold color) and Na_v1.5-E1784K (PDB ID: 7DTC, transparent magenta) displays a lateral dilation of the VSDs. The inset shows the dilation of the PD. (B) Comparative analysis of the conformation of GC residues of individual VSDs in Model-II, Na_v1.5-E1784K (PDB ID: 7DTC, magenta), and rNa_v1.5c/QQQ (PDB ID: 7FBS, cyan). GC residues are shown in stick representation. An1 and An2 denote anion1 and anion2, respectively. OR denotes the occluding residue. For clarity, only the S2 and S4 segments of all the VSDs are shown. (C) Superimposition of Model-I (blue), Model-II (green), and Na_v1.5-E1784K (magenta). The III-IV linker and its connecting S0_IV helix are highlighted and labeled. The conformational changes of the IFM and III-IV linker helix are shown in the upper right inset. The lower inset shows the electrostatic surface potential of the IFM receptor bound to the IFM motif of Model-II. The IFM residues in the receptor are highlighted. (D) Overall structural alignment between Model-II (green) and Na_v1.5-E1784K (magenta). Conserved residues involved in hydrophobic interactions at the IFM receptor of Model-II (green) and Na_v1.5-E1784K (magenta) are shown in bold stick. The IFM residues in the receptor are highlighted. (E) Conserved residues involved in polar interactions at the IFM receptor after overall structural alignment. D1484 moves downward from Na_v1.5-E1784K (magenta) to Model-II (green). D1484 and K1492 form a salt bridge in Model-II. The IFM residues in the receptor are highlighted. (F) Interaction between the IFM motif and the receptor pocket residues after structural alignment. The side chain of F1473 has moved downward which causes displacement of the IFM motif. Key residues are shown in stick representation. The IFM residues in the receptor are highlighted. (G) The III-IV linker and outward tilting of the S0_IV helix are highlighted in the overlay of Model-I, Model-II, and Na_v1.5-E1784K. (H) The translation of the flexible loop of the III-IV linker is associated with the tilting of the S0_IV helix. The Cα sphere represents the positions of three mutational hotspot residues.

Fig. 3.

Conformational dynamics of the CTD, kinetic analysis, and the comparison of the activation gate in the open state. (A) Superimposition of Model-I (blue) and Model-II (green). The positions of the III-IV linker and CTD are highlighted. (B) The position of the CTD differs by > 9° between Model-I and Model-II. (C) Key residues K1504 and K1505 of the III-IV linker are near the negatively charged surface of the CTD (Left). K1504 of the III-IV linker interacts with E1867 in the CTD. K1505 of the III-IV linker interacts with E1788 in the CTD of Model-II (Right). (D) Electrophysiological recordings of current–voltage relationships displayed a faster time course of inactivation for K1504E, K1505E, and E1867K. (E) Charge-reversal mutants K1504E, K1505E, E1788K, and E1867K cause a hyperpolarized shift in steady-state inactivation. A depolarized shift in the conductance curve was seen for K1504E and E1867K. (F) E1867K displayed a slower recovery from inactivation. (G) Comparison of the activation gate diameter in Model-I, Model-II, Na_v1.5-E1784K (PDB ID: 7DTC), and rNa_v1.5c/QQQ (PDB ID: 7FBS). The black and orange dashed lines represent the diameter at the Top and Bottom layers of the activation gate, respectively. (H) The permeation paths of Model-I and Model-II are shown as gray dots. SF: selectivity filter, CC: central cavity, AG: activation gate. (I) The corresponding pore radii are compared with that of Na_v1.5-E1784K and rNa_v1.5c/QQQ.

Fig. 4.

MD analysis of Model-II and structural mapping of mutations linked to BrS and LQT3. (A) Average hydration along the pore axis at different TMV. At lower TMV (~100 mV), the AG region is dehydrated. At higher TMV (>350 mV), pore hydration is increased (dashed, solid blue, and red curve) at the AG. The dotted cyan line indicates bulk water density. Solid and dashed curves represent hydration profiles at positive and negative TMV, respectively. (B) Hydration profile along the pore axis at 0 mV and −713 mV TMV for Model-II during the 600 ns MD simulation. (C) Snapshots of a Na⁺ (black sphere) permeation event during MD simulation. SF: selectivity filter, CC: central cavity, AG: activation gate. (D) Structural mapping of mutation hotspot residues (sphere). (E) A cluster of selected mutations associated with BrS and LQT3 in the region of the III-IV linker and CTD. (F) Interaction between R1512 and F1522 in Model-I (blue), Model-II (green), and Na_v1.5-E1784K (magenta). The π-cation interaction occurs exclusively in Model-II. (G) E1867K mutation presented a significant increase in persistent current, but no change in persistent current was observed in K1504E, K1505E, and E1788K.

Fig. 5.

Mechanism of fast inactivation. Transition from the open state to the inactivated state. The conformational changes in each state are assigned with letters. (a) S2_II and S4_IV sequentially move upward, transitioning from a partially depolarized to a fully depolarized conformation. (b) Overall dilation is reduced. (c) Then the S0_IV helix is slanted outward and forms an extended conformation of the flexible loop of the III–IV linker. (d) S4–S5_IV linker and III-IV linker helices move upward. (e) IFM motif undergoes a transition from a loosely bound state to a semitight conformation. (f) The CTD partially moves away from the III-IV linker, resulting in a loss of electrostatic interactions. Transition from the intermediate state to inactivated state: (g) A further reduction in overall dilation. (h) S0_IV is slanted inward and forms a relaxed conformation of the flexible loop of the III-IV linker. (i) S4–S5_IV linker and III-IV linker helices move further upward. (j) IFM motif undergoes a shift from a semitight conformation to a tightly bound state. (k) The CTD moves further away and retains a dynamic conformation. Schematics are not drawn to scale.