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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

The influence of β1- and β3-subunits on Nav1.7 voltage-dependence of activation and inactivation. (A–D) Normalized currents for both activation and steady-state inactivation (see Materials and Methods) are plotted against the test potential. Each panel compares Nav1.7 alone to Nav1.7 co-expressed with individual β-subunits. A minor but significant effect on the V½of activation was observed for β3-subunit (n = 17, hyperpolarizing shift, p < 0.05) co-expression as compared to Nav1.7 alone (n = 81). β1- (n = 39), β2- (n = 25) and β4-subunits (n = 13) did not modify the V½of activation of Nav1.7. The effect on the V½ of inactivation was highly significant for β1- (n = 51, depolarizing shift with p < 0.0001) and β3-subunit (n = 18, depolarizing shift with p < 0.0001) compared to Nav1.7 alone (n = 92); whereas β2- (n = 43) and β4-subunits (n = 20) had no effect. Individual points are the mean ± s.e.m. of the normalized current at each voltage point. The smooth curves are Boltzmann fits whose equations give both the V½of activation and inactivation (midpoints) and their associated slope factors (see Materials and Methods). The V½ of steady-state activation and inactivation comparing Nav1.7 alone vs. the co-expression with each subunit were tested by One-Way ANOVA followed by Bonferroni's multiple comparison tests. Values and statistics can be found in Table 1.
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Figure 2: The influence of β1- and β3-subunits on Nav1.7 voltage-dependence of activation and inactivation. (A–D) Normalized currents for both activation and steady-state inactivation (see Materials and Methods) are plotted against the test potential. Each panel compares Nav1.7 alone to Nav1.7 co-expressed with individual β-subunits. A minor but significant effect on the V½of activation was observed for β3-subunit (n = 17, hyperpolarizing shift, p < 0.05) co-expression as compared to Nav1.7 alone (n = 81). β1- (n = 39), β2- (n = 25) and β4-subunits (n = 13) did not modify the V½of activation of Nav1.7. The effect on the V½ of inactivation was highly significant for β1- (n = 51, depolarizing shift with p < 0.0001) and β3-subunit (n = 18, depolarizing shift with p < 0.0001) compared to Nav1.7 alone (n = 92); whereas β2- (n = 43) and β4-subunits (n = 20) had no effect. Individual points are the mean ± s.e.m. of the normalized current at each voltage point. The smooth curves are Boltzmann fits whose equations give both the V½of activation and inactivation (midpoints) and their associated slope factors (see Materials and Methods). The V½ of steady-state activation and inactivation comparing Nav1.7 alone vs. the co-expression with each subunit were tested by One-Way ANOVA followed by Bonferroni's multiple comparison tests. Values and statistics can be found in Table 1.

Mentions: Whether the INa density increase mediated by both β1- and β3-subunits was also accompanied by alterations of other Nav1.7 biophysical properties was also assessed. The voltage dependence of macroscopic INa activation and inactivation (see Materials and Methods) of Nav1.7 in the absence and presence of each β-subunit was recorded and analyzed. The co-transfection of the β1-subunit significantly shifted the V½ of steady-state inactivation toward depolarized potentials by ~5.8 mV, but had no influence on V½ of activation (Figure 2A and Table 1). The β3-subunit shifted the V½ of inactivation toward depolarized potentials by ~3.5 mV and the V½ of activation toward hyperpolarized potentials by ~3.7 mV (Figure 2C and Table 1). Neither the β2- nor β4-subunits affected Nav1.7 voltage dependence of activation or inactivation (Figures 2B,D and Table 1).


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)

The influence of β1- and β3-subunits on Nav1.7 voltage-dependence of activation and inactivation. (A–D) Normalized currents for both activation and steady-state inactivation (see Materials and Methods) are plotted against the test potential. Each panel compares Nav1.7 alone to Nav1.7 co-expressed with individual β-subunits. A minor but significant effect on the V½of activation was observed for β3-subunit (n = 17, hyperpolarizing shift, p < 0.05) co-expression as compared to Nav1.7 alone (n = 81). β1- (n = 39), β2- (n = 25) and β4-subunits (n = 13) did not modify the V½of activation of Nav1.7. The effect on the V½ of inactivation was highly significant for β1- (n = 51, depolarizing shift with p < 0.0001) and β3-subunit (n = 18, depolarizing shift with p < 0.0001) compared to Nav1.7 alone (n = 92); whereas β2- (n = 43) and β4-subunits (n = 20) had no effect. Individual points are the mean ± s.e.m. of the normalized current at each voltage point. The smooth curves are Boltzmann fits whose equations give both the V½of activation and inactivation (midpoints) and their associated slope factors (see Materials and Methods). The V½ of steady-state activation and inactivation comparing Nav1.7 alone vs. the co-expression with each subunit were tested by One-Way ANOVA followed by Bonferroni's multiple comparison tests. Values and statistics can be found in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The influence of β1- and β3-subunits on Nav1.7 voltage-dependence of activation and inactivation. (A–D) Normalized currents for both activation and steady-state inactivation (see Materials and Methods) are plotted against the test potential. Each panel compares Nav1.7 alone to Nav1.7 co-expressed with individual β-subunits. A minor but significant effect on the V½of activation was observed for β3-subunit (n = 17, hyperpolarizing shift, p < 0.05) co-expression as compared to Nav1.7 alone (n = 81). β1- (n = 39), β2- (n = 25) and β4-subunits (n = 13) did not modify the V½of activation of Nav1.7. The effect on the V½ of inactivation was highly significant for β1- (n = 51, depolarizing shift with p < 0.0001) and β3-subunit (n = 18, depolarizing shift with p < 0.0001) compared to Nav1.7 alone (n = 92); whereas β2- (n = 43) and β4-subunits (n = 20) had no effect. Individual points are the mean ± s.e.m. of the normalized current at each voltage point. The smooth curves are Boltzmann fits whose equations give both the V½of activation and inactivation (midpoints) and their associated slope factors (see Materials and Methods). The V½ of steady-state activation and inactivation comparing Nav1.7 alone vs. the co-expression with each subunit were tested by One-Way ANOVA followed by Bonferroni's multiple comparison tests. Values and statistics can be found in Table 1.
Mentions: Whether the INa density increase mediated by both β1- and β3-subunits was also accompanied by alterations of other Nav1.7 biophysical properties was also assessed. The voltage dependence of macroscopic INa activation and inactivation (see Materials and Methods) of Nav1.7 in the absence and presence of each β-subunit was recorded and analyzed. The co-transfection of the β1-subunit significantly shifted the V½ of steady-state inactivation toward depolarized potentials by ~5.8 mV, but had no influence on V½ of activation (Figure 2A and Table 1). The β3-subunit shifted the V½ of inactivation toward depolarized potentials by ~3.5 mV and the V½ of activation toward hyperpolarized potentials by ~3.7 mV (Figure 2C and Table 1). Neither the β2- nor β4-subunits affected Nav1.7 voltage dependence of activation or inactivation (Figures 2B,D and Table 1).

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