Computational Investigation of Voltage-Gated Sodium Channel β3 Subunit Dynamics

Abstract

Voltage-gated sodium (Na _v ) channels form the basis for the initiation of the action potential in excitable cells by allowing sodium ions to pass through the cell membrane. The Na _v channel α subunit is known to function both with and without associated β subunits. There is increasing evidence that these β subunits have multiple roles that include not only influencing the voltage-dependent gating but also the ability to alter the spatial distribution of the pore-forming α subunit. Recent structural data has shown possible ways in which β1 subunits may interact with the α subunit. However, the position of the β1 subunit would not be compatible with a previous trimer structure of the β3 subunit. Furthermore, little is currently known about the dynamic behavior of the β subunits both as individual monomers and as higher order oligomers. Here, we use multiscale molecular dynamics simulations to assess the dynamics of the β3, and the closely related, β1 subunit. These findings reveal the spatio-temporal dynamics of β subunits and should provide a useful framework for interpreting future low-resolution experiments such as atomic force microscopy.

Keywords: coarse-grain; epilepsy; lipid bilayer; molecular dynamics; multiscale.

FIGURE 1

Orientations of the β1/3 subunit on the membrane. (A) The Na_v β1 subunit complex, highlighting the orientation, and interaction of the β1 subunit with respect to the membrane [PDB: 6AGF (Pan et al., 2018)]. (B) Structure of the trimeric Ig domain from β3 [PDB: 4L1D (Namadurai et al., 2014)]. (C) Overlay of the trimeric β3 Ig domain on the Ig domain of the β1 subunit, demonstrating the anticipated position of the β1 TMD and suggesting that these conformations are not compatible. The approximate location of the membrane is indicated by a gray box and dotted lines.

FIGURE 2

Pitch angles of the β1/3 Ig domain. (A) The starting conformation of both β1 and β3 subunits. (B) Schematic illustrating the pitch angle, θp (see section “Ig Orientation Analysis” for precise definition). (C) Histogram of the pitch angles visited of over 400 ns × 25 runs of the β1 – subunit. (D) Histogram of the pitch angles visited of over 400 ns × 25 runs of the β3 – subunit. (E) Heatplot of pitch angles over 400 ns in the β1 subunit. (F) Heatplot of pitch angles over 400 ns in the β3 subunit.

FIGURE 3

Tilting and position of E177(β1)/176(β3) in the β subunit transmembrane domain. (A) Histogram of TMD tilt angles over 25 × 400 ns simulations of the β1 (red) and β3 (blue) subunits. (B) Schematic of the angle used to measure the tilt angle in the TMD, phosphorus atoms of the POPC bilayer are shown as orange spheres. (C) Histogram of minimum distances between E177 (β1)/E176 (β3) (center of mass of sidechain oxygens) and the nitrogen atom of the surrounding POPC headgroups over 25 × 400 ns. The shoulder at a distance of 7 Å reflects the initial starting coordinates. (D) Position in the membrane of the conserved glutamic acid residue (highlighted inside a red box) in the β3 subunit after 400 ns. (E) Closer look at E176 (β3) in (D) with two nearby POPC residues interacting with the terminal oxygen atoms of the residue.

FIGURE 4

Regions of high interaction on the β subunit with a POPC membrane and non-conserved residues between subunits. (A) Regions of frequent interaction on the β1 subunit. (B) Regions of high interaction on the β3 subunit. The ECD of β1 exhibits more frequent regions of contact when compared to β3. (C) Non-conserved residues (shown as gray spheres) in the β1 subunit Ig and linker domains (some labels omitted in right hand image for clarity).

FIGURE 5

Ig domain dynamics in the mutated β1 subunit. Pitch angle analysis of the (A) β1 Ig_mut, (B) β1 Ig_mut + linker_mut, and (C) β1 linker_mut. Snapshots of conformations for each system are shown with mutated domains indicated in red.

FIGURE 6

Trimeric model of full-length β3. (A) Shows the initial configuration with TM helices that almost parallel to the membrane normal. During the simulation, the TM helices adopt tilted orientations and the ECD domain continues to sit in a similar position to the initial configuration. (B) Average Cα RMSDs (from three runs) for the whole trimer (blue), the TM domains (orange, residues F153 to E189 of each subunit), the ECD only (green). Pale background reflects one standard deviation. (C) Distribution of tilt angles for the three helices in the trimer. (D) Probability density colored from white to red to black mapped onto the structure to show the lipid-protein interactions. (E) shows the key residues of the ECD that form interactions with the membrane. In both (D,E) different protein monomers are indicated in superscript.

FIGURE 7

β3 clustering in a general mammalian membrane. (A) Regions of high protein - protein contact visualized on the β3 subunit surface colored as a probability undergoing an interaction with another protein. Spheres indicate residues with total interactions above 2.5% of the total time. (B) Typical clustering of β3 subunits (green) in a mixed lipid membrane (viewed from the extracellular side). Lipids visible in the upper leaflet include POPC (gray), POPE (green), Sph (pink), GM3 (purple), and Chol (orange). (C) Evolution of β3 clusters over a 10 μs simulation. Lighter colors indicate higher order clusters. (D) Distinct conformations of the β3 subunit involved in clusters. From left to right: down, intermediate, and up states.

FIGURE 8

CG β3 interactions in a mixed lipid membrane. (A) Protein – lipid interactions visualized on face 1 of the β3 model. The first side chain particle of residues that account for over 2.5% of the total interaction time are shown as spheres. (B) Radial distribution function of the distribution of lipids surrounding β3 subunits in the PM.