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 potentials¹. Aberrations in their function are associated with a wide spectrum of cardiac diseases including arrhythmias and heart failure^2-5. Despite decades of progress in Na_v1.5 biology^6-8, the lack of structural insights into intracellular regions has hampered our understanding of its gating mechanisms. Here we present three cryo-EM structures of human Na_v1.5 in previously unanticipated 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 the IFM motif repositioned in the receptor site but not dislodged. In particular, 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 results in the 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.

Figure 1. Cryo-EM structures of full-length hNa_v1.5.

a, Side (left) and intracellular (right) view of the cryo-EM reconstruction of Model-I. Individual domains and inter-domain linkers are segmented and color-coded. The lower panel depicts the atomic structure of Model-I including the resolved inter-domain linkers, lipid molecules, the cholesterol plug, 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 of Model-II. Color-coded according to (a). The CTD is segmented and shown in pink. The lower panel shows the atomic structure of Model II. c, Side view of the cryo-EM reconstruction (top) and atomic structure (bottom) of Model-III. Color-coded according to (b).

Figure 2. Insights into the pore domains, VSDs, and III-IV linkers of hNa_v1.5.

a, The intracellular view of the structural superimposition of Model-I (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-III, Na_v1.5-E1784K, and rNa_v1.5c/QQQ (PDB ID: 7FBS). GC residues are shown in stick representation. An1 and An2 denote anion1 and anion2, respectively. OR is occluding residue. For clarity, only the S2 and S4 segments of all the VSDs are shown. c, Superimposition of Model-I, Model-II, Model-III, and Na_v1.5-E1784K (magenta). The III-IV linker and its connecting S0_IV helix are highlighted. The conformational changes of the IFM and III-IV linker helix are shown in the upper right inset. The lower inset displays the electrostatic surface potential of the IMF receptor bound to the IFM motif of Model-III. d, Hydrophobic interactions at the IMF receptor of Model-I (bright orange), Model-III (splitpea), and Na_v1.5-E1784K (teal). e, Polar interactions at the IMF receptor. D1484 moves downward from NaV1.5-E1784K to Model-III. D1484 and K1492 form a salt bridge in Model-I. f, Interaction between the IFM motif and the receptor pocket residues. Key residues are shown in stick representation.

Figure 3. Molecular interactions near the IFM motif, as well as the conformational dynamics of III-IV linker and CTD.

a, The III-IV linker and outward tilting of the S0_IV helix are highlighted in the overlay of Model-I, Model-II, Model-III, and Na_v1.5-E1784K. b, The translation of the flexible loop of the III-IV linker is associated with the tilting of the S0_IV helix. The sphere represents the positions of three mutational hotspot residues. c, Positioning of the III-IV linker and CTD in Model-II and Model-III. d, The position of the CTD differs by > 9° between Model II and Model III. e, Key residues, K1504 and K1505, of the III-IV linker are in proximity to the negatively charged surface of CTD (left) and near to E1867 and E1788 residues of the CTD of Model-III (right). f, Electrophysiological recordings of current-voltage relationships displayed a faster time course of inactivation for K1504E, K1505E, and E1867K. g, 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. h, E1867K displayed a slower recovery from inactivation.

Figure 4. Activation gate diameter in open state.

a, Comparison of the activation gate diameter in Model-I, Model-II, and Model-III, Na_v1.5-E1784K (PDB: 7DTC), and rNa_v1.5c/QQQ (PDB: 7FBS). The black and orange dashed lines represent the diameter at the top and bottom layers of the activation gate, respectively. b, The permeation paths of Model-I and Model-III are shown as grey dots. SF: selectivity filter, CC: central cavity, AG: activation gate. c, The corresponding pore radii are compared with that of Na_v1.5-E1784K and rNa_v1.5c/QQQ.

Figure 5. Structural mapping of mutations linked to BrS and LQT3.

a, Structural mapping of mutation hotspot residues (sphere). b, A cluster of selected mutations associated with BrS and LQT3 in the region of III-IV linker and CTD. c, Interaction between R1512 and F1522 in Model-I (bright orange), Model-II (marine), and Model-III (split pea). The π-cation interaction occurs exclusively in Model-III. d,E1867K mutation presented a significant increase in persistent current, but no change in persistent current was observed in K1504E, K1505E, and E1788K.

Figure 6. 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 semi-tight 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 semi-tight conformation to a tightly bound state. (k) CTD moves further away and retains a dynamic conformation. Schematics are not drawn to scale.