GPD1L links redox state to cardiac excitability by PKC-dependent phosphorylation of the sodium channel SCN5A

Abstract

The SCN5A-encoded cardiac sodium channel underlies excitability in the heart, and dysfunction of sodium current (I(Na)) can cause fatal ventricular arrhythmia in maladies such as long QT syndrome, Brugada syndrome (BrS), and sudden infant death syndrome (SIDS). The gene GPD1L encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1), but the function for this enzyme is unknown. Mutations in GPD1L have been associated with BrS and SIDS and decrease I(Na) through an unknown mechanism. Using a heterologous expression system, we show that GPD1L associated with SCN5A and that the BrS- and SIDS-related mutations in GPD1L caused a loss of enzymatic function resulting in glycerol-3-phosphate PKC-dependent phosphorylation of SCN5A at serine 1503 (S1503) through a GPD1L-dependent pathway. The direct phosphorylation of S1503 markedly decreased I(Na). These results show a function for GPD1L in cell physiology and a mechanism linking mutations in GPD1L to sudden cardiac arrest. Because the enzymatic step catalyzed by GPD1L depends upon nicotinamide adenine dinucleotide, this GPD1L pathway links the metabolic state of the cell to I(Na) and excitability and may be important more generally in cardiac ischemia and heart failure.

Fig. 1.

Glycerol 3-phosphate dehydrogenase 1-like (GPD1L) mutations decreased sodium current (I_Na) density mediated by PKC phosphorylation at serine 1503 (S1503). A–D: representative traces for families of macroscopic I_Na measured from human embryonic kidney (HEK293) cells coexpressing wild-type (WT) and mutant constructs of cardiac sodium channel α-subunit (SCN5A) and GPD1L as indicated. The cells were depolarized for 24 ms to different potentials in increments of 10 mV from a holding potential of −140 mV (see protocol inset) in control conditions and after 3–5 h incubation with 1-oleoyl-2-acetylglycerol (OAG; 20 μM), glycerol-3-phosphate (G3P; 10 μM), and G3P plus staurosporine (Sta; 1 μM) before patch-clamp recordings. E: summary data for I_Na density measurements at −20 mV from a holding potential of −140 mV for SCN5A with GPD1L-WT and mutants (n = 5–32). S/A, SCN5A-S1503A *P < 0.05 vs. GPDlL-WT control (Con) and vs. GPD1L-WT/SCN5A-S1503A; #P < 0.05 vs. GPD1L-mutant/SCN5A-WT in Con.

Fig. 2.

Enzymatic activity of GPD1L decreased by the GPD1L missense mutations E83K and A280V. A: G3P oxidation was measured spectrophotometrically at 37°C from the WT and glutathione S-transferase (GST)-labeled GPD1L proteins cleaved by factor Xa to be separated from GST-tag (materials and methods; n = 6 in each group). B: diagram of proposed GPD1L-PKC pathway with substrates and pharmacological blockers (indicated by T-shaped lines) that affect SCN5A-GPD1L association via PKC activation. DAG, diacylglycerol; PA, phosphatidic acid; LysoPA, lsophoshpatidic acid; DHAP, dihydroxyacetone phosphate. *P < 0.05 vs. GPD1L WT.

Fig. 3.

GPD1L-PKC stimulation through the G3P pathway decreased I_Na density for SCN5A with GPD1L-WT. A: summary data for I_Na density at −20 mV from a holding potential of −140 mV for SCN5A with GPD1L-WT after treatment with various pathway substrates and inhibitors: 10 μM G3P, 300 μM NADH, 10 μM OAG, 100 μM quecertin (Que), 300 μM NAD, 10 μM propranolol (Pro), 1 μM staurosporine (Sta), and 10 μM thimerosol (Thi). For columns 5–8, the treatment was with 2 compounds as indicated by the labels with a column, with G3P present or NADH present. For GPD1L-E83K (dark gray) and GPD1L-A280V (light gray), recordings were performed after incubation with 100 μM Pro added 3–5 h before patch-clamp recording. The dotted lines represent the I_Na density in GPD1L-WT for control conditions and in G3P from Fig. 1E (n = 5–23). *P < 0.05 vs. control; #P < 0.05 vs. G3P, Que, or NADH alone. B: acidosis decreased I_Na density through a PKC-dependent mechanism. HEK293 cells were transfected with SCN5A-WT and GPD1L-WT and incubated overnight at pH of 7.4 or pH of 6.5 before transfer and recording of I_Na in control (normal pH) bath solution. The dotted line represents the value for SCN5A-WT + GPD1L-WT at pH 7.4 from Fig. 1E. The I_Na-attenuating effects of acidic pH with GPD1L-WT were prevented by both Sta and Pro (n = 6–12). *P < 0.05 vs. control at pH 7.4; #P < 0.05 vs. control at pH 6.5.

Fig. 4.

Flowcytometric analysis of cell surface expression of the sodium channel complex. HEK293 cells transiently transfected with SCN5A, GPD1L, and FLAG-SCN1B were harvested (see materials and methods), and cell surface staining was performed by FITC-conjugated anti-FLAG M2 antibody, without permeabilization. Gray areas reflect signals from cells expressing SCN5A, GPD1L, and FLAG-SCN1B constructs, and the black line indicates signals from SCN5A, GPD1L, and SCN1B without FLAG as negative control. In this method, the count of cells with surface expression is reflected in the long tail rather than the peak. Marker 1 (M1) was established at a point containing 5% of negative control. The numbers above M1 represent the percentage of positive events falling within the M1 region and reflect cell surface expression. A and B: cell surface expression of the SCN5A. GPD1L complex was measured from HEK cells coexpressing SCN5A, SCN1B, and GPD1L-WT under control conditions (A) and after 3–5 h incubation with OAG (20 μM), or G3P (10 μM) and G3P plus staurosporine (1 μM) 1 μM stausporin (B), or with GPD1L-E83K or GPD1L-A280V (C and D). GPD1L-E83K and -A280V decreased cell surface expression, and this effect was reversed by staurosporine and by expression of SCN5A with a mutated PKC phosphorylation site (SCN5A-S1503A; C and D). E: summary data for I_Na density measurements shown in A–D. The data represent the percent ratio with the minimal (26.4%) surface expression obtained by testing SCN5A-G1743R, an established trafficking defective mutation (Ref. 20) (data not shown) and maximal (40.1%) surface expression seen in control conditions.

Fig. 5.

GPD1L associated with SCN5A and GPD1L mutations do not disrupt this association. HEK cells stably expressing SCN5A were transfected with GST constructs of GPD1L-WT, E83K, or A280V and lysed 48 h after transfection. Cell lysates were examined for protein interaction using the pull-down approach. GST-GPD1L-bound proteins were analyzed by SDS-PAGE and immunoblotted with anti-cardiac sodium channel antibody.