Modulation of Pore Opening of Eukaryotic Sodium Channels by π-Helices in S6

Abstract

Voltage-gated sodium channels are heterotetrameric sodium selective ion channels that play a central role in electrical signaling in excitable cells. With recent advances in structural biology, structures of eukaryotic sodium channels have been captured in several distinct conformations corresponding to different functional states. The secondary structure of the pore lining S6 helices of subunits DI, DII, and DIV has been captured with both short π-helix stretches and in fully α-helical conformations. The relevance of these secondary structure elements for pore gating is not yet understood. Here, we propose that a π-helix in at least DI-S6, DIII-S6, and DIV-S6 results in a fully conductive state. On the other hand, the absence of π-helix in either DI-S6 or DIV-S6 yields a subconductance state, and its absence from both DI-S6 and DIV-S6 yields a nonconducting state. This work highlights the impact of the presence of a π-helix in the different S6 helices of an expanded pore on pore conductance, thus opening new doors toward reconstructing the entire conformational landscape along the functional cycle of Nav Channels and paving the way to the design of state-dependent modulators.

Figure 1

Architecture of eukaryotic Nav channels. A. Eukaryotic Nav channels exist as a single polypeptide chain arranged as a tetramer. B. Top and C. side view of the experimentally resolved structure of cardiac sodium channel (Nav1.5, PDB: 7FBS). Inset shows a zoomed-in view of the bound inactivation particle (IFM) interacting with the conserved N1767 residue in subunit IV. D. The pore lined by S6 helices can be divided into three main regions: selectivity filter, central cavity, and activation gate. E. Top view of the pore of the experimental open state structure. The cartoon representation shows a simplified version of the structure. This representation will be used throughout this article. Pentagons represent S6s containing π-helices, and squares represent fully α-helical S6s.

Figure 2

The presence of a π-helix in DI-S6 and DIV-S6 is important for ion conduction. A. Cartoon representation of the different pore conformations. B. Pore hydration around the activation gate in the different pore conformations. C. Hydrophobic residues lining the pore in the 1pi_d3 model (green, 0 ns), 2pi_d1_d3 (purple, 100 ns), 2pi_d3_d4 (gray, 43 ns), and 2pi_d2_d3 (blue, 42 ns). D. Ion permeation free energy profiles in the models with a hydrated pore (2pi_d1_d3 in purple and 2pi_d3_d4 in gray). E. Average conductance values in 2pi_d1_d3 (purple) and 2pi_d3_d4 (gray) from simulations. The blue dashed lines show the experimental conductance values for the first and second open state. Error bars represent the standard error across six replicates. P-value < 0.05 indicates that there is a significant difference in the means between the two different models while a p-value > 0.05 indicates that there is no significant difference.

Figure 3

The presence of a π-helix in at least DI-S6, DIII-S6 and DIV-S6 is sufficient to generate a fully conductive pore. A. Cartoon representation of the different pore conformations. B. Pore hydration around the activation gate in the different pore conformations (Black: 3pi_d2_d3_d4, Brown: 3pi_d1_d2_d3, Orange: 3pi_d1_d3_d4, Red: 4pi). C. Average conductance values in the different models across six replicates from simulations. The blue dashed lines show the experimental conductance values for the first and second open state. Error bars represent the standard error across six replicates. p-value < 0.05 indicates that there is a significant difference in the means between the two different models while a p-value > 0.05 indicates that there is no significant difference. D. Ion permeation free energy profile in the different models

Figure 4

Flowchart showing the effect of removing/adding π-helices to different S6 helices. The orange-shaded region shows the pore conformations that have a fully conductive pore. The blue-shaded region shows the nonconductive pore conformations. The gray-shaded region shows pore conformations that are sub conductive. The pentagons represent π-helices and the squares represent α-helices in the S6 helix. The pentagon following a tick mark shows that a π-helix in the corresponding S6 can affect ion permeation, while a cross mark signifies that it does not affect ion permeation.