Mechanistic insights into the interaction of the MOG1 protein with the cardiac sodium channel Nav1.5 clarify the molecular basis of Brugada syndrome

Abstract

Na_v1.5 is the α-subunit of the cardiac sodium channel complex. Abnormal expression of Na_v1.5 on the cell surface because of mutations that disrupt Na_v1.5 trafficking causes Brugada syndrome (BrS), sick sinus syndrome (SSS), cardiac conduction disease, dilated cardiomyopathy, and sudden infant death syndrome. We and others previously reported that Ran-binding protein MOG1 (MOG1), a small protein that interacts with Na_v1.5, promotes Na_v1.5 intracellular trafficking to plasma membranes and that a substitution in MOG1, E83D, causes BrS. However, the molecular basis for the MOG1/Nav1.5 interaction and how the E83D substitution causes BrS remains unknown. Here, we assessed the effects of defined MOG1 deletions and alanine-scanning substitutions on MOG1's interaction with Na_v1.5. Large deletion analysis mapped the MOG1 domain required for the interaction with Na_v1.5 to the region spanning amino acids 146-174, and a refined deletion analysis further narrowed this domain to amino acids 146-155. Site-directed mutagenesis further revealed that Asp-148, Arg-150, and Ser-151 cluster in a peptide loop essential for binding to Na_v1.5. GST pulldown and electrophysiological analyses disclosed that the substitutions E83D, D148Q, R150Q, and S151Q disrupt MOG1's interaction with Na_v1.5 and significantly reduce its trafficking to the cell surface. Examination of MOG1's 3D structure revealed that Glu-83 and the loop containing Asp-148, Arg-150, and Ser-151 are spatially proximal, suggesting that these residues form a critical binding site for Na_v1.5. In conclusion, our findings identify the structural elements in MOG1 that are crucial for its interaction with Na_v1.5 and improve our understanding of how the E83D substitution causes BrS.

Keywords: Brugada syndrome (BrS); MOG1; Nav1.5 sodium channel; SCN5A; arrhythmia; cardiac disease; cardiovascular disease; genetic disorder; membrane trafficking; molecular modeling; myocardial ischemia; protein-protein interaction; sodium channel; trafficking.

Figure 1.

Large deletion analysis of MOG1 to identify the Na_v1.5 interaction domain. A, diagram (dark, alpha helix; gray, beta strand) showing WT MOG1 (MOG1-WT) and mutant MOG1 with three serial deletions from the C terminus (MOG1–174, MOG1–146, and MOG1–101). B, raw traces of sodium current recorded from Na_v1.5/HEK cells transfected with WT MOG1, each deletion mutant or vector negative control pcDNA3.1- (−). C, effect of MOG1-WT and each deletion mutant on sodium current as compared with vector control. Error bars indicate S.E. D, peak sodium current density (pA/pF). The peak current density (pA/pF) was −234.49 ± 100.74 for pcDNA 3.1- (−) (20 cells), −342.55 ± 117.29 for MOG1-WT (20 cells), −347.41 ± 202.50 for MOG1–174 (18 cells), −245.64 ± 106.86 for MOG1–146 (19 cells), and −237.38 ± 114.13 for MOG1–101 (18 cells). Values are mean ± S.D.

Figure 2.

Interaction between WT and mutant MOG1 and Na_v1.5. A, the tsA201 cells were transfected with an expression plasmid for FLAG-tagged WT MOG1 (MOG1-WT) or each deletion mutant, lysed and mixed with GST–Na_v1.5–LII with the intracellular loop II of Na_v1.5 or control GST alone purified by GSH-Sepharose from Escherichia coli transformed with respective GST-plasmids. GST pulldown was then carried out, and MOG1 was detected using Western blot analysis with an anti-FLAG antibody. B, quantified data on the relative binding levels between WT MOG1, mutant MOG1, and Na_v1.5-LII from studies as in A. Error bars indicate S.D. (n = 3).

Figure 3.

Microdeletion analysis of MOG1 for amino acid residues from 174 to 126 to identify the Na_v1.5 interaction domain. A, diagram showing WT MOG1 (MOG1-WT) and mutant MOG1 with five serial deletions from residues 174 to 126. B, effect of MOG1-WT and each deletion mutant on sodium current as compared with vector control pcDNA3.1- (−). Error bars indicate S.E. C, peak sodium current density (pA/pF). The peak current density (pA/pF) was −212.56 ± 55.43 for pcDNA3.1- (−) (11 cells), −330.30 ± 111.08 for MGO1-WT (13 cells), −331.01 ± 125.37 for MOG1-Δ166–174 (12 cells), −343.54 ± 156.59 for MOG1-Δ156–165 (12 cells), −199.49 ± 84.01 for MOG1-Δ146–155 (11 cells), −212.31 ± 98.83 for MOG1-Δ136–145 (11 cells), and −335.72 ± 165.29 for MOG1-Δ126–135 (11 cells). Values are mean ± S.D.

Figure 4.

Interaction between WT and mutant MOG1 and each deletion mutant in a region from 126 to 174 and Na_v1.5. A, GST pulldown assays revealed that MOG1-Δ146–155 and MOG1-Δ136–145 failed to interact with GST–Na_v1.5–LII. MOG1-Δ166–174, MOG1-Δ156–165, and MOG1-Δ126–135 were successfully pulled down by GST–Na_v1.5–LII. B, quantified data on the relative binding levels between WT and mutant MOG1 and Na_v1.5–LII from studies as in A. Error bars indicate S.D. (n = 3).

Figure 5.

Identification of the critical amino acid residues for MOG1-Na_v1.5 function. Alanine-scanning mutagenesis was used to systematically mutate each amino acid residue between 136 and 155. A, effect of WT MOG1 (MOG1-WT) and each point mutation on the peak sodium current density. The peak current density (pA/pF) was −214.73 ± 122.06 for pcDNA3.1- (−) (18 cells), −335.46 ± 146.39 for MOG1-WT (22 cells), −323.56 ± 177.54 for T136A (18 cells), −339.94 ± 215.48 for D137A (21 cells), −231.09 ± 103.11 for L138A (26 cells), −211.92 ± 128.54 for L139A (18 cells), −339.44 ± 158.05 for L140A (21 cells), −342.86 ± 141.42 for T141A (20 cells), −340.58 ± 180.76 for F142A (24 cells), −328.57 ± 194.45 for N143A (20 cells), −382.30 ± 232.42 for Q144A (22 cells), −349.78 ± 180.99 for P145A (22 cells), −317.06 ± 128.00 for P146A (16 cells), −341.18 ± 162.71 for P147A (16 cells), −252.88 ± 94.36 for D148A (26 cells), −309.47 ± 134.74 for N149A (19 cells), −244.82 ± 113.98 for R150A (27 cells), −230.35 ± 116.52 for S151A (24 cells), −316.84 ± 124.46 for S152A (18 cells), −321.18 ± 141.01 for L153A (20 cells), −328.34 ± 170.50 for G154A (20 cells), and −322.94 ± 146.94 for P155A (17 cells). Values are mean ± S.D. B, GST pulldown assays. MOG1 mutations L138A or L139A impaired the interaction between MOG1 and GST–Na_v1.5–LII and failed to increase sodium current density. Mutations D148A, R150A, or S151A significantly decreased MOG1 interaction with GST–Na_v1.5–LII and the peak sodium current density. C, quantified data on the relative binding levels between WT and mutant MOG1 and Na_v1.5–LII from studies as in B. Error bars indicate S.D. (n = 3). D, diagram showing the location of residues Glu-83, Leu-138, Leu-139, Asp-148, Arg-150, Ser-151 in the structure of MOG1. The MOG1 structure was from Protein Data Bank (PDB ID: 5YFG).

Figure 6.

Amino acid residues Asp-148, Arg-150, and Ser-151 of MOG1 are critical for interaction with Na_v1.5 and for increasing sodium current density. Residues Asp-148, Arg-150, and Ser-151 were mutated to glutamine with a predicted larger effect than alanine. A, effect of WT MOG1 and mutant D148Q, R150Q, or S151Q on sodium current density. Error bars indicate S.E. B, effect of WT MOG1 and mutant D148Q, R150Q, or S151Q on peak sodium current density. The peak current density (pA/pF) was −237.18 ± 153.35 for pcDNA3.1- (−) (8 cells), −381.73 ± 136.76 for MOG1-WT (12 cells), −227.89 ± 136.40 for D148Q (9 cells), −246.88 ± 123.32 for R150Q (13 cells), and −219.10 ± 121.21 for S151Q (8 cells). Values are mean ± S.D. C, GST pulldown assays for interaction between GST–Na_v1.5–LII and D148Q, R150Q, S151Q, and WT MOG1. D, quantified data on the relative binding levels between WT and mutant MOG1 and Na_v1.5–LII from studies as in C. Error bars indicate S.D. (n = 3).

Figure 7.

MOG1 mutants L138A, L139A, D148Q, R150Q, and S151Q all failed to increase Na_v1.5 expression in the plasma membrane (PM). A and C, Western blotting images of Na_v1.5 in the plasma membranes and in the total lysates from HEK293 cells co-transfected with pcDNA-SCN5A and WT MOG1 or mutant MOG1. B and D, quantification of plasma membrane (PM) and total protein expression levels of Na_v1.5 in A and C, respectively. Error bars indicate S.D. (n = 3).

Figure 8.

Brugada syndrome associated MOG1 mutation E83D disrupts the interaction between MOG1 and Na_v1.5 and reduces Na_v1.5 levels at cell surface. A, GST pulldown assays. GST–Na_v1.5–LII pulled WT MOG1 down, but not mutant MOG1 with mutation E83D. Error bars indicate S.D. (n = 3). B, Western blotting images showing Na_v1.5 expression levels in plasma membranes and total cell lysates. C, quantification of plasma membrane (PM) Na_v1.5 expression levels and total Na_v1.5 expression levels from images in B. Error bars indicate S.D. (n = 3).

Figure 9.

MOG1 mutations D148Q, R150Q, and S151Q that disrupt the interaction between MOG1 and Na_v1.5 act in a dominant negative manner as Brugada syndrome mutation E83D. A, effects of MOG1 mutations E83D, D148Q, R150Q, and S151Q on sodium current densities in tsA201 cells co-transfected with 0.2 μg of pcDNA3-SCN5A and an equal amount of WT MOG1 (MOG1-WT). The empty vector (1 μg) was used as a negative control, and MOG1-WT (1 μg) and MOG1-WT (0.5 μg) + vector (0.5 μg) were used as positive controls. Error bars indicate S.E. B, the peak sodium current densities (pA/pF) derived from A: −68.93 ± 32.05 for vector pcDNA3.1- (−) (24 cells), −124.14 ± 43.46 for MOG1-WT (1 μg) (17 cells), −98.20 ± 23.43 for MOG1-WT (0.5 μg) + vector (0.5 μg) (16 cells), −73.62 ± 37.79 for MOG1-WT (0.5 μg) + D148Q (0.5 μg) (23 cells), −72.06 ± 40.67 for MOG1-WT (0.5 μg) + R150Q (0.5 μg) (22 cells), −73.59 ± 40.41 for MOG1-WT (0.5 μg) + Ser-151 (0.5 μg) (25 cells), and −74.77 ± 35.14 for MOG1-WT (0.5 μg) + E83D (0.5 μg) (25 cells). Values are mean ± S.D. C, GST pulldown assays for interaction between GST–Na_v1.5–LII and WT MOG1, and an equal amount of WT MOG1 and each mutant MOG1 (D148Q, R150Q, S151Q, or E83D). Note that more DNA was needed for transfection for GST pulldown assays than for I_Na recording. D, quantified data from Western blot analysis as in (C) for the relative binding levels between GST–Na_v1.5–LII and WT MOG1, and WT/mutants. Error bars indicate S.D. (n = 3).