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β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells.

Laedermann CJ, Syam N, Pertin M, Decosterd I, Abriel H - Front Cell Neurosci (2013)

Bottom Line: Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties.The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa.This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

View Article: PubMed Central - PubMed

Affiliation: Pain Center, Department of Anesthesiology, University Hospital Center and University of Lausanne Lausanne, Switzerland ; Department of Clinical Research, University of Bern Bern, Switzerland.

ABSTRACT
Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V ½ of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

No MeSH data available.


Related in: MedlinePlus

β-subunits regulate Nav1.7 currents. (A) Typical whole-cell Na+ currents of HEK293 cells transfected with Nav1.7 alone or Nav1.7 co-expressed with individual β-subunits elicited with a typical current-voltage protocol. (B) Voltage-dependence of current decay of Nav1.7 alone compared to Nav1.7 with each individual β-subunit. Inset: Normalized representative current traces of Nav1.7 elicited by test pulses at 0 mV. Co-transfection of the β1- (p = 0.011, n = 39), β2- (p < 0.0001, n = 25), β3- (p < 0.0001, n = 16), and β4-subunit (p = 0.006, n = 13) decreased the time constant decay as compared to Nav1.7 alone (n = 81). Two-Ways ANOVA and Bonferroni post-hoc tests. Data are expressed as mean ± s.e.m. (C)INa densities from HEK293 cells transfected with Nav1.7 alone or co-transfected with individual β-subunits. β1- (n = 55) and β3-subunits (n = 34), but not β2- (n = 67) nor β4-subunits (n = 27), increased the Nav1.7 current densities. p < 0.0001 for β1- and β3-subunits with One-Way ANOVA followed by Bonferroni post-hoc tests. Data are expressed as mean ± s.e.m. and were normalized to Nav1.7 alone for each experiments. Values can be found in Table 1. *p < 0.05, **p < 0.01 and ***p < 0.001.
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Figure 1: β-subunits regulate Nav1.7 currents. (A) Typical whole-cell Na+ currents of HEK293 cells transfected with Nav1.7 alone or Nav1.7 co-expressed with individual β-subunits elicited with a typical current-voltage protocol. (B) Voltage-dependence of current decay of Nav1.7 alone compared to Nav1.7 with each individual β-subunit. Inset: Normalized representative current traces of Nav1.7 elicited by test pulses at 0 mV. Co-transfection of the β1- (p = 0.011, n = 39), β2- (p < 0.0001, n = 25), β3- (p < 0.0001, n = 16), and β4-subunit (p = 0.006, n = 13) decreased the time constant decay as compared to Nav1.7 alone (n = 81). Two-Ways ANOVA and Bonferroni post-hoc tests. Data are expressed as mean ± s.e.m. (C)INa densities from HEK293 cells transfected with Nav1.7 alone or co-transfected with individual β-subunits. β1- (n = 55) and β3-subunits (n = 34), but not β2- (n = 67) nor β4-subunits (n = 27), increased the Nav1.7 current densities. p < 0.0001 for β1- and β3-subunits with One-Way ANOVA followed by Bonferroni post-hoc tests. Data are expressed as mean ± s.e.m. and were normalized to Nav1.7 alone for each experiments. Values can be found in Table 1. *p < 0.05, **p < 0.01 and ***p < 0.001.

Mentions: The functional impact of the co-expression of the four β-subunits on Nav1.7-mediated INa was studied by performing whole cell patch-clamp experiments in HEK293 cells. Each β-subunit was independently co-transfected with Nav1.7 and then compared to Nav1.7 expressed alone. Figure 1A shows typical traces of Nav1.7 INa obtained with a current-voltage protocol. A hastening of the Nav1.7 current decay kinetics was observed with each of the β-subunits tested (Figure 1B). The shortening of the Nav1.7 time constant of current decay was observed for a wide range of voltages and showed voltage-dependency for every β-subunit (Figure 1B). The shortening was particularly prominent for the β3-subunit. In addition, β1- and β3-subunits also significantly increased (~2-fold) Nav1.7-mediated current density as compared to Nav1.7 alone or to Nav1.7 co-expressed with β2- or β4-subunits (Figure 1C and Table 1). We also observed that β2 and β4-subunits did not antagonize β1 and β3-subunits-dependent up-regulation, and that the two latter have additive positive effect on Nav1.7-mediated current (data not shown).


β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells.

Laedermann CJ, Syam N, Pertin M, Decosterd I, Abriel H - Front Cell Neurosci (2013)

β-subunits regulate Nav1.7 currents. (A) Typical whole-cell Na+ currents of HEK293 cells transfected with Nav1.7 alone or Nav1.7 co-expressed with individual β-subunits elicited with a typical current-voltage protocol. (B) Voltage-dependence of current decay of Nav1.7 alone compared to Nav1.7 with each individual β-subunit. Inset: Normalized representative current traces of Nav1.7 elicited by test pulses at 0 mV. Co-transfection of the β1- (p = 0.011, n = 39), β2- (p < 0.0001, n = 25), β3- (p < 0.0001, n = 16), and β4-subunit (p = 0.006, n = 13) decreased the time constant decay as compared to Nav1.7 alone (n = 81). Two-Ways ANOVA and Bonferroni post-hoc tests. Data are expressed as mean ± s.e.m. (C)INa densities from HEK293 cells transfected with Nav1.7 alone or co-transfected with individual β-subunits. β1- (n = 55) and β3-subunits (n = 34), but not β2- (n = 67) nor β4-subunits (n = 27), increased the Nav1.7 current densities. p < 0.0001 for β1- and β3-subunits with One-Way ANOVA followed by Bonferroni post-hoc tests. Data are expressed as mean ± s.e.m. and were normalized to Nav1.7 alone for each experiments. Values can be found in Table 1. *p < 0.05, **p < 0.01 and ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757325&req=5

Figure 1: β-subunits regulate Nav1.7 currents. (A) Typical whole-cell Na+ currents of HEK293 cells transfected with Nav1.7 alone or Nav1.7 co-expressed with individual β-subunits elicited with a typical current-voltage protocol. (B) Voltage-dependence of current decay of Nav1.7 alone compared to Nav1.7 with each individual β-subunit. Inset: Normalized representative current traces of Nav1.7 elicited by test pulses at 0 mV. Co-transfection of the β1- (p = 0.011, n = 39), β2- (p < 0.0001, n = 25), β3- (p < 0.0001, n = 16), and β4-subunit (p = 0.006, n = 13) decreased the time constant decay as compared to Nav1.7 alone (n = 81). Two-Ways ANOVA and Bonferroni post-hoc tests. Data are expressed as mean ± s.e.m. (C)INa densities from HEK293 cells transfected with Nav1.7 alone or co-transfected with individual β-subunits. β1- (n = 55) and β3-subunits (n = 34), but not β2- (n = 67) nor β4-subunits (n = 27), increased the Nav1.7 current densities. p < 0.0001 for β1- and β3-subunits with One-Way ANOVA followed by Bonferroni post-hoc tests. Data are expressed as mean ± s.e.m. and were normalized to Nav1.7 alone for each experiments. Values can be found in Table 1. *p < 0.05, **p < 0.01 and ***p < 0.001.
Mentions: The functional impact of the co-expression of the four β-subunits on Nav1.7-mediated INa was studied by performing whole cell patch-clamp experiments in HEK293 cells. Each β-subunit was independently co-transfected with Nav1.7 and then compared to Nav1.7 expressed alone. Figure 1A shows typical traces of Nav1.7 INa obtained with a current-voltage protocol. A hastening of the Nav1.7 current decay kinetics was observed with each of the β-subunits tested (Figure 1B). The shortening of the Nav1.7 time constant of current decay was observed for a wide range of voltages and showed voltage-dependency for every β-subunit (Figure 1B). The shortening was particularly prominent for the β3-subunit. In addition, β1- and β3-subunits also significantly increased (~2-fold) Nav1.7-mediated current density as compared to Nav1.7 alone or to Nav1.7 co-expressed with β2- or β4-subunits (Figure 1C and Table 1). We also observed that β2 and β4-subunits did not antagonize β1 and β3-subunits-dependent up-regulation, and that the two latter have additive positive effect on Nav1.7-mediated current (data not shown).

Bottom Line: Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties.The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa.This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

View Article: PubMed Central - PubMed

Affiliation: Pain Center, Department of Anesthesiology, University Hospital Center and University of Lausanne Lausanne, Switzerland ; Department of Clinical Research, University of Bern Bern, Switzerland.

ABSTRACT
Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V ½ of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

No MeSH data available.


Related in: MedlinePlus