Isoform-specific N-linked glycosylation of NaV channel α-subunits alters β-subunit binding sites

Abstract

Voltage-gated sodium channel α-subunits (NaV1.1-1.9) initiate and propagate action potentials in neurons and myocytes. The NaV β-subunits (β1-4) have been shown to modulate α-subunit properties. Homo-oligomerization of β-subunits on neighboring or opposing plasma membranes has been suggested to facilitate cis or trans interactions, respectively. The interactions between several NaV channel isoforms and β-subunits have been determined using cryogenic electron microscopy (cryo-EM). Interestingly, the NaV cryo-EM structures reveal the presence of N-linked glycosylation sites. However, only the first glycan moieties are typically resolved at each site due to the flexibility of mature glycan trees. Thus, existing cryo-EM structures may risk de-emphasizing the structural implications of glycans on the NaV channels. Herein, molecular modeling and all-atom molecular dynamics simulations were applied to investigate the conformational landscape of N-linked glycans on NaV channel surfaces. The simulations revealed that negatively charged sialic acid residues of two glycan sites may interact with voltage-sensing domains. Notably, two NaV1.5 isoform-specific glycans extensively cover the α-subunit region that, in other NaV channel α-subunit isoforms, corresponds to the binding site for the β1- (and likely β3-) subunit immunoglobulin (Ig) domain. NaV1.8 contains a unique N-linked glycosylation site that likely prevents its interaction with the β2 and β4-subunit Ig-domain. These isoform-specific glycans may have evolved to facilitate specific functional interactions, for example, by redirecting β-subunit Ig-domains outward to permit cis or trans supraclustering within specialized cellular compartments such as the cardiomyocyte perinexal space. Further experimental work is necessary to validate these predictions.

Figure 1.

Na_V channel α-subunit domains and β-subunits. (A) Key structural features of the Na_V channel α-subunit. The four internally homologous domains, DI–IV, are color-coded and labeled. Within DI, the transmembrane helices, S1–6 (including the positively-charged S4 helix), the voltage-sensing domain (VSD), and the S5–6 extracellular turret loop (ECTL) are labeled. (B) Cryo-EM structure of human Na_V1.5 in top and side view (PDB: 7DTC). (C) The resolved binding sites of β1 (magenta), β2 (cyan), and β4 (yellow) based on cryo-EM structures of human Na_V1.1 (PDB: 7DTD); Na_V1.2 (PDB: 6J8E) and Na_V1.4 (PDB: 6AGF), as depicted from top and side views.

Figure 2.

Position and structural representation of Na_V glycans. (A) The N-linked glycan positions on Na_V channel cryo-EM structures are listed in a table format. Each color denotes homologous N-linked glycosylation sites within the indicated α-subunit isoform. (B) The representative N-linked glycan tree used in this study is shown in 2D format. (C) The corresponding 3D representation of the glycan tree is shown in B. N-acetylglucosamines are shown in blue, mannose in green, galactose in yellow, fucose in red, and sialic acid in pink. Connectivity (alpha = A, beta = B) between glycan moieties is notated (B).

Figure 3.

N-linked glycans on Na_V channels. (A) Alignments of the amino acid sequences of the Na_V1.1–1.9 domain I ECTLs are shown. The residues are colored by similarity (yellow), conservation among 50% of sequences or higher (green), or 100% conservation (orange letters and in green blocks). All NX[S or T] motifs in the sequence alignments are outlined with red rectangles to note the evolutionary conservation of the motifs. (B and C) The conserved glycans on the domain I ECTLs are colored crimson and shown on the modeled Na_V1.5 structure. In C, the unique Na_V1.5 glycans above VSD III are shown in crimson. (D and E) Alignments of the amino acid sequences of the Na_V1.1–1.9 domain II and III, respectively. Residue conservation and NX[S or T] motifs are colored as in A. (F) The unique Na_V1.8 N-linked glycan N819 is shown in crimson on the modeled Na_V1.8 structure. (G) The conserved glycans on domain III are colored in crimson and shown on the modelled Na_V1.5 structure.

Figure 4.

N-linked glycan density on Na _V 1.4 and Na _V 1.5. Images show overlapping and superimposed snapshots of glycan conformations extracted from the molecular dynamics simulations every 5 ns, over a time frame of 150 ns. (A) Top view of Na_V1.4. (B and C) Side views of Na_V1.4, VSDs IV and II, respectively. (D) Top view of Na_V1.5. (E and F) Side views of Na_V1.5, VSDs IV and II, respectively. Glycans interacting with VSD IV are depicted. The Na_V1.4 (C) and Na_V1.5 (F) glycans interacting near VSD II are displayed. Glycans are shown in dark gold, with sialylated tips in red.

Figure 5.

Effect of N-linked glycan density on β-subunit binding for Na _V 1.4 and Na _V 1.5. Images show overlapping and superimposed snapshots of glycan conformations from the molecular dynamics simulations, extracted every 5 ns, over a time frame of 150 ns. (A) Top view of Na_V1.4, with β1 (magenta), β2 (cyan), and β4 (yellow). (B) Side view of Na_V1.4 interacting with β2 and β4. (C) Side view of Na_V1.4 interacting with β1. (D) Top view of Na_V1.5, with β1, β2, and β4. (E) Side view of Na_V1.5 interacting with β2, and β4, assuming the same binding as with Na_V1.4. (F) Side view of Na_V1.5 interacting with β1, assuming the same binding as with Na_V1.4. Glycans are shown in dark gold, with sialylated tips in red.

Figure 6.

Putative trans interactions between Na _V 1.5/β1 complexes. (A and B) Full snapshots (A) and close-up (B) image of the “tip-to-tip” models of the trans interactions between Na_V1.5/β1 complexes on opposing membranes (grey). (C and D) Full snapshots and (D) close-up image of the “side-to-side” models. Note the potential for glycan hindrance in the side-to-side model.

Figure 7.

Mutations at N-linked glycan sites linked to clinical pathologies. (A) The N-linked glycan sites disrupted by mutations and their associated pathologies are listed. (B–D) The N-linked glycans affected by mutations (orange) in (B) Na_V1.1; (C) Na_V1.5 and (D) Na_V1.6 are shown amongst the other N-linked glycans (grey) on the Na_V channel surfaces.