Structural basis of cytoplasmic NaV1.5 and NaV1.4 regulation

Abstract

Voltage-gated sodium channels (NaVs) are membrane proteins responsible for the rapid upstroke of the action potential in excitable cells. There are nine human voltage-sensitive NaV1 isoforms that, in addition to their sequence differences, differ in tissue distribution and specific function. This review focuses on isoforms NaV1.4 and NaV1.5, which are primarily expressed in skeletal and cardiac muscle cells, respectively. The determination of the structures of several eukaryotic NaVs by single-particle cryo-electron microscopy (cryo-EM) has brought new perspective to the study of the channels. Alignment of the cryo-EM structure of the transmembrane channel pore with x-ray crystallographic structures of the cytoplasmic domains illustrates the complementary nature of the techniques and highlights the intricate cellular mechanisms that modulate these channels. Here, we review structural insights into the cytoplasmic C-terminal regulation of NaV1.4 and NaV1.5 with special attention to Ca2+ sensing by calmodulin, implications for disease, and putative channel dimerization.

Figure 1.

Schematic of Na_Vs. (a) The Na_Vs are pseudotetramers with four transmembrane domains (DI–DIV, purple), with each domain containing six α-helices (S1–S6). The cytoplasmic CT has been shown to have six α-helices (αI–αVI; pink) with αI–αV forming the EFL. (b) The open configuration of the transmembrane domains, with S1–S6 of one domain shaded in purple to show S1–S4 (dark purple) forming the VSD that shifts to open the pore formed by S5–S6 (light purple).

Figure 2.

Published crystal structures of CTNa_V with CaM. (a) Structures aligned pairwise to CTNa_V1.5–CaM (PDB accession no. 4OVN) using the EFL domain as an anchor. Each CT is in shades of red, CaM in green, and FHF in blue. The angle of helix αVI with respect to the EFL varies in these constructs. (b) The same structures aligned pairwise using the CaM C-lobe as an anchor and so helix αVI has the same orientation. These alignments highlight the different relative orientations of the EFL and helix αVI, displaying the EFL to the right (4OVN, 6MBA, 6MC9) or to the left (4DCK, 4JPZ, 4JQ0, 6MUD). (c) Complexes of CaM with shorter peptides of CTNa_V1.5.

Figure 3.

Cryo-EM structures of Na_Vs. α-Subunits are shown in purple and β-subunits are shown in yellow. 5X0M displays most of the N-terminal domain and the EFL of the CT domain.

Figure 4.

Alignment of transmembrane Na_VPas with CTNa_V1.5. (a) Na_VPas (PDB accession no. 5X0M) with cytoplasmic domain resolution. DIII–DIV linker is shown in cyan and EFL of CT in purple. (b) CTNa_V1.5 is shown in pink and apo-CaM in green (PDB accession no. 4OVN). (c) Alignment of Na_VPas with CTNa_V1.5 shows overlap between the two structures (0.39 RMSD over 69 amino acids).

Figure 5.

Na_V1.4 displays CDI. (a) Na_V1.5 Na⁺ current does not change upon Ca²⁺ release, but Na_V1.4 I_Na is reduced upon optical Ca²⁺ release, indicated with a blue line. (b) When CTNa_V1.4 is transplanted to Na_V1.5 and vice versa, the CDI still only takes place in channels with CTNa_V1.4. (c) Deletion of the Na_V1.5 post-IQ motif reveals CDI with I_Na reduced at 10 µM Ca²⁺. a and b are adapted from Ben-Johny et al. (2014), and c is adapted from Yoder et al. (2019).

Figure 6.

Populations of CTNa_V1.4 and CTNa_V1.5 with bound CaM. (a) Panels show the relative population (z-axis) of four CaM species bound to CTNa_V, as a function of Ca²⁺ and CaM concentration. Black boxes indicate the dominant species at high CaM and 10 µM Ca²⁺; (Ca²⁺)_2C–CaM for CTNa_V1.4 (top) and (Ca²⁺)₄–CaM for CTNa_V1.5 (bottom). (b) Cross-section showing populations of CTNa_V (Ca²⁺)–CaM species as a function of [Ca²⁺] at a [CaM] of 10 µM. Note the dramatic reduction of (Ca²⁺)_2C–CaM for CTNa_V1.5 compared with CTNa_V1.4. Adapted from Yoder et al. (2019).

Figure 7.

Sequence alignment of the post-IQ sequence in the Na_V1s. (a) Alignment of the IQ and the post-IQ domains of the nine major human Na_V isoforms. (b) Logo of the post-IQ sequences where the frequency of an amino acid at a given position is proportional to the size of its single amino acid letter descriptor.

Figure 8.

Mutations in Na_V1.5 mapped to its structure. (a) Overall structure of Na_V1.5 with the transmembrane portion as observed in the cryo-EM structure (PDB accession no. 6UZ3) in purple. CT domain as observed in PDB accession no. 4OVN, shown in pink surface with mutations displayed in red. (b) Zoom-in showing mutations in the EFL and IQ region, potentially interacting with (or in close proximity to) CaM. (c) Mutations interacting with FHF of PDB accession no. 4JQ0. (d) Mutations of Na_V1.5 in the EFL that line the binding site of the DIII–DIV linker.

Figure 9.

Mutations in Na_V1.4 mapped to its structure. (a) Overall structure of Na_V1.4 with the transmembrane portion as observed in the cryo-EM structure (PDB accession no. 6AGF) in purple and Na_V β1 in yellow. CT domain as observed in PDB accession no. 6MBA, shown in orange surface with mutations displayed in white. (b) Different 6MBA Na_V1.4 orientation with FHF (from PDB accession no. 4JQ0 of Na_V1.5 with FHF1b; aligned to 6MBA by CTNa_V EFL) to show a possible interaction with Q1633.

Figure 10.

Na_V–Na_V dimerization: interaction between CT helix αVI and EFL of an adjacent channel. (a) Front view of the complex of CTNa_V1.5–CaM (red surface) with another channel’s helix αVI (pink ribbons) as observed in PDB accession no. 4OVN. CaM bound to each channel is shown in shades of green. (b) Bird’s eye view of a; the binding site of helix αVI of channel 1 (pink ribbons) on EFL of channel 2 (red surface). (c) Rotated 90° with both channels 1 and 2 shown in ribbons. Na_V1.5 channel 1 is shown in pink with its CaM (light green), and Na_V1.5 channel 2 EFL in red with its CaM (dark green).