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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

β1- and β3-subunit mediate differential forms of Nav1.7 whose expression is increased at the membrane. (A) Representative western blot of a biotinylation assay with total lysate (input, left) and cell surface (biotinylation, right) fractions from HEK293 cells transiently transfected with Nav1.7 alone, or co-expressed with each individual β-subunit and the associated quantifications. Input: Nav1.7 is detected in two forms: a fast migrating band (~250 kDa, that will be referred to as lower band) that consist mostly of the Nav1.7 immunoreactive signal and a slow migrating band (~280 kDa, that will be referred to as upper band). β1- (p = 0.006), β2- (p = 0.003), and β4-subunits (p = 0.009) significantly decreased Nav1.7 expression, whereas the β3-subunit had no effect (p = 0.570). Because the upper band was below the sensitivity threshold, both bands were quantified together. Biotinylation: Nav1.7 membrane protein is detected in three forms. When expressed alone, one lower band (white triangle, ~250 kDa) and one upper band (black triangle, ~280 kDa) were present (for identification of these bands, see Panel B). When the β1-subunit is co-expressed, the upper band was clearly shifted into an intermediate migrating band (~260 kDa) with increased expression (p = 0.047). β2- and β4-subunits revealed the same pattern as when Nav1.7 was transfected alone and did not change its expression, except for the small decrease of the lower band when the β4-subunit is co-transfected (p = 0.020). The β3-subunit clearly increased Nav1.7 immunoreactivity of the lower band (p < 0.0001). For input and biotinylation fractions, actin and the α1-subunit of NaK-ATPase were used as biotin leakiness and loading controls, respectively. Data represent mean ± s.e.m, n = 4 independent experiments. Student's unpaired t-test, each condition being compared with Nav1.7. *p < 0.05, **p < 0.01 and ***p < 0.001. (B) Representative western blot and identification of glycosylation state of Nav1.7 in biotinylated fraction from HEK293 cells transiently transfected with Nav1.7. EndoH only cleaves the lower band of biotinylated Nav1.7, demonstrating that this band represents the core-glycosylated form of the channel. The upper band is digested by PNGaseF, demonstrating that it corresponds to fully-glycosylated form of the channel. PNGaseF can also digest the core-glycosylated form of Nav1.7.
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Figure 5: β1- and β3-subunit mediate differential forms of Nav1.7 whose expression is increased at the membrane. (A) Representative western blot of a biotinylation assay with total lysate (input, left) and cell surface (biotinylation, right) fractions from HEK293 cells transiently transfected with Nav1.7 alone, or co-expressed with each individual β-subunit and the associated quantifications. Input: Nav1.7 is detected in two forms: a fast migrating band (~250 kDa, that will be referred to as lower band) that consist mostly of the Nav1.7 immunoreactive signal and a slow migrating band (~280 kDa, that will be referred to as upper band). β1- (p = 0.006), β2- (p = 0.003), and β4-subunits (p = 0.009) significantly decreased Nav1.7 expression, whereas the β3-subunit had no effect (p = 0.570). Because the upper band was below the sensitivity threshold, both bands were quantified together. Biotinylation: Nav1.7 membrane protein is detected in three forms. When expressed alone, one lower band (white triangle, ~250 kDa) and one upper band (black triangle, ~280 kDa) were present (for identification of these bands, see Panel B). When the β1-subunit is co-expressed, the upper band was clearly shifted into an intermediate migrating band (~260 kDa) with increased expression (p = 0.047). β2- and β4-subunits revealed the same pattern as when Nav1.7 was transfected alone and did not change its expression, except for the small decrease of the lower band when the β4-subunit is co-transfected (p = 0.020). The β3-subunit clearly increased Nav1.7 immunoreactivity of the lower band (p < 0.0001). For input and biotinylation fractions, actin and the α1-subunit of NaK-ATPase were used as biotin leakiness and loading controls, respectively. Data represent mean ± s.e.m, n = 4 independent experiments. Student's unpaired t-test, each condition being compared with Nav1.7. *p < 0.05, **p < 0.01 and ***p < 0.001. (B) Representative western blot and identification of glycosylation state of Nav1.7 in biotinylated fraction from HEK293 cells transiently transfected with Nav1.7. EndoH only cleaves the lower band of biotinylated Nav1.7, demonstrating that this band represents the core-glycosylated form of the channel. The upper band is digested by PNGaseF, demonstrating that it corresponds to fully-glycosylated form of the channel. PNGaseF can also digest the core-glycosylated form of Nav1.7.

Mentions: Whether the Nav1.7-mediated INa upregulation could be due to an increase of Nav1.7 protein density at the cell membrane was investigated by performing biotinylation of plasma membrane proteins. After lysis, proteins were sampled under reducing conditions known to dissociate the covalently bound β2- and β4-subunits from α-subunits (Messner and Catterall, 1985). Co-transfection of β1-, β2-, and β4-subunits significantly decreased Nav1.7 protein expression in the total cell lysate fraction (input, Figure 5A). The quantification revealed a ~2-fold decrease for each of these three subunits. Co-transfection of the β3-subunit had no effect on Nav1.7 expression in the total cell lysate fraction. In the biotinylated membrane fraction two bands at different apparent molecular weights were observed when Nav1.7 was expressed alone (white and black arrow heads in Figure 5A). These bands correspond to different glycosylated states of Nav1.7 as demonstrated by using deglycosylating enzymes (Figure 5B). Endoglycosidase H (EndoH) only cleaves core N-glycans from proteins whereas Peptide-N-Glycosidase F (PNGaseF) does not discriminate between full and core glycosylated proteins. Of the two bands of biotinylated Nav1.7, only the lower was sensitive to EndoH and was shifted to an apparent lower molecular weight band (compare white arrowhead in the first lane to gray arrowhead in the third lane), indicating that this band corresponds to the core-glycosylated form of the channel (Figure 5B). Because PNGaseF was able to digest both bands, it can be proposed that the higher band corresponds to the fully-glycosylated form of Nav1.7. The lower band of biotinylated Nav1.7 migrates at the same apparent molecular weight as the band observed in the input fraction (white arrow heads in Figure 5A) suggesting that most of Nav1.7 in the intracellular pool is core-glycosylated. This is consistent with the channel being early and rapidly, but only partially, glycosylated after its synthesis. The upper band in the total cell lysate fraction was faint and blurry (Figure 5A), suggesting that the fully-glycosylated channel only represents a small fraction of the total Nav1.7 cellular pool. It was only by enriching the membrane proteins through the precipitation of the biotinylated membrane fraction (the ratio between the amount of lysate protein loaded and the amount of streptavidin beads needed to precipitate biotinylated proteins was ~1:30) that the upper band was distinctly observed. Co-expression of the β1-subunit reproducibly shifted the upper band to an intermediate migrating band of lower apparent molecular weight. This suggests that the β1-subunit mediates an alternative glycosylated form of Nav1.7. When comparing the β1-subunit-modified intermediate band with the upper band of the control condition (Nav1.7 alone), a significant increase in signal intensity was observed (Figure 5A, quantification), which is consistent with the increase in the Nav1.7 current density (Figure 1C). Co-transfection of the β2-subunit neither modified the glycosylation pattern nor the expression of any of the two bands, consistent with the fact that the current density was not modified. β3-subunit expression also altered the Nav1.7 band pattern in the biotinylated fractions. The upper band overlapped with the lower band under the migrating conditions used. The β3-subunit significantly increased (~7-fold) the intensity of the lower band as compared to the lower band of control, consistent with the increase of the Nav1.7 current density elicited by the β3-subunit (Figure 1C). Finally, β4-subunit co-transfection led to a small but significant decrease of the lower band.


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)

β1- and β3-subunit mediate differential forms of Nav1.7 whose expression is increased at the membrane. (A) Representative western blot of a biotinylation assay with total lysate (input, left) and cell surface (biotinylation, right) fractions from HEK293 cells transiently transfected with Nav1.7 alone, or co-expressed with each individual β-subunit and the associated quantifications. Input: Nav1.7 is detected in two forms: a fast migrating band (~250 kDa, that will be referred to as lower band) that consist mostly of the Nav1.7 immunoreactive signal and a slow migrating band (~280 kDa, that will be referred to as upper band). β1- (p = 0.006), β2- (p = 0.003), and β4-subunits (p = 0.009) significantly decreased Nav1.7 expression, whereas the β3-subunit had no effect (p = 0.570). Because the upper band was below the sensitivity threshold, both bands were quantified together. Biotinylation: Nav1.7 membrane protein is detected in three forms. When expressed alone, one lower band (white triangle, ~250 kDa) and one upper band (black triangle, ~280 kDa) were present (for identification of these bands, see Panel B). When the β1-subunit is co-expressed, the upper band was clearly shifted into an intermediate migrating band (~260 kDa) with increased expression (p = 0.047). β2- and β4-subunits revealed the same pattern as when Nav1.7 was transfected alone and did not change its expression, except for the small decrease of the lower band when the β4-subunit is co-transfected (p = 0.020). The β3-subunit clearly increased Nav1.7 immunoreactivity of the lower band (p < 0.0001). For input and biotinylation fractions, actin and the α1-subunit of NaK-ATPase were used as biotin leakiness and loading controls, respectively. Data represent mean ± s.e.m, n = 4 independent experiments. Student's unpaired t-test, each condition being compared with Nav1.7. *p < 0.05, **p < 0.01 and ***p < 0.001. (B) Representative western blot and identification of glycosylation state of Nav1.7 in biotinylated fraction from HEK293 cells transiently transfected with Nav1.7. EndoH only cleaves the lower band of biotinylated Nav1.7, demonstrating that this band represents the core-glycosylated form of the channel. The upper band is digested by PNGaseF, demonstrating that it corresponds to fully-glycosylated form of the channel. PNGaseF can also digest the core-glycosylated form of Nav1.7.
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Figure 5: β1- and β3-subunit mediate differential forms of Nav1.7 whose expression is increased at the membrane. (A) Representative western blot of a biotinylation assay with total lysate (input, left) and cell surface (biotinylation, right) fractions from HEK293 cells transiently transfected with Nav1.7 alone, or co-expressed with each individual β-subunit and the associated quantifications. Input: Nav1.7 is detected in two forms: a fast migrating band (~250 kDa, that will be referred to as lower band) that consist mostly of the Nav1.7 immunoreactive signal and a slow migrating band (~280 kDa, that will be referred to as upper band). β1- (p = 0.006), β2- (p = 0.003), and β4-subunits (p = 0.009) significantly decreased Nav1.7 expression, whereas the β3-subunit had no effect (p = 0.570). Because the upper band was below the sensitivity threshold, both bands were quantified together. Biotinylation: Nav1.7 membrane protein is detected in three forms. When expressed alone, one lower band (white triangle, ~250 kDa) and one upper band (black triangle, ~280 kDa) were present (for identification of these bands, see Panel B). When the β1-subunit is co-expressed, the upper band was clearly shifted into an intermediate migrating band (~260 kDa) with increased expression (p = 0.047). β2- and β4-subunits revealed the same pattern as when Nav1.7 was transfected alone and did not change its expression, except for the small decrease of the lower band when the β4-subunit is co-transfected (p = 0.020). The β3-subunit clearly increased Nav1.7 immunoreactivity of the lower band (p < 0.0001). For input and biotinylation fractions, actin and the α1-subunit of NaK-ATPase were used as biotin leakiness and loading controls, respectively. Data represent mean ± s.e.m, n = 4 independent experiments. Student's unpaired t-test, each condition being compared with Nav1.7. *p < 0.05, **p < 0.01 and ***p < 0.001. (B) Representative western blot and identification of glycosylation state of Nav1.7 in biotinylated fraction from HEK293 cells transiently transfected with Nav1.7. EndoH only cleaves the lower band of biotinylated Nav1.7, demonstrating that this band represents the core-glycosylated form of the channel. The upper band is digested by PNGaseF, demonstrating that it corresponds to fully-glycosylated form of the channel. PNGaseF can also digest the core-glycosylated form of Nav1.7.
Mentions: Whether the Nav1.7-mediated INa upregulation could be due to an increase of Nav1.7 protein density at the cell membrane was investigated by performing biotinylation of plasma membrane proteins. After lysis, proteins were sampled under reducing conditions known to dissociate the covalently bound β2- and β4-subunits from α-subunits (Messner and Catterall, 1985). Co-transfection of β1-, β2-, and β4-subunits significantly decreased Nav1.7 protein expression in the total cell lysate fraction (input, Figure 5A). The quantification revealed a ~2-fold decrease for each of these three subunits. Co-transfection of the β3-subunit had no effect on Nav1.7 expression in the total cell lysate fraction. In the biotinylated membrane fraction two bands at different apparent molecular weights were observed when Nav1.7 was expressed alone (white and black arrow heads in Figure 5A). These bands correspond to different glycosylated states of Nav1.7 as demonstrated by using deglycosylating enzymes (Figure 5B). Endoglycosidase H (EndoH) only cleaves core N-glycans from proteins whereas Peptide-N-Glycosidase F (PNGaseF) does not discriminate between full and core glycosylated proteins. Of the two bands of biotinylated Nav1.7, only the lower was sensitive to EndoH and was shifted to an apparent lower molecular weight band (compare white arrowhead in the first lane to gray arrowhead in the third lane), indicating that this band corresponds to the core-glycosylated form of the channel (Figure 5B). Because PNGaseF was able to digest both bands, it can be proposed that the higher band corresponds to the fully-glycosylated form of Nav1.7. The lower band of biotinylated Nav1.7 migrates at the same apparent molecular weight as the band observed in the input fraction (white arrow heads in Figure 5A) suggesting that most of Nav1.7 in the intracellular pool is core-glycosylated. This is consistent with the channel being early and rapidly, but only partially, glycosylated after its synthesis. The upper band in the total cell lysate fraction was faint and blurry (Figure 5A), suggesting that the fully-glycosylated channel only represents a small fraction of the total Nav1.7 cellular pool. It was only by enriching the membrane proteins through the precipitation of the biotinylated membrane fraction (the ratio between the amount of lysate protein loaded and the amount of streptavidin beads needed to precipitate biotinylated proteins was ~1:30) that the upper band was distinctly observed. Co-expression of the β1-subunit reproducibly shifted the upper band to an intermediate migrating band of lower apparent molecular weight. This suggests that the β1-subunit mediates an alternative glycosylated form of Nav1.7. When comparing the β1-subunit-modified intermediate band with the upper band of the control condition (Nav1.7 alone), a significant increase in signal intensity was observed (Figure 5A, quantification), which is consistent with the increase in the Nav1.7 current density (Figure 1C). Co-transfection of the β2-subunit neither modified the glycosylation pattern nor the expression of any of the two bands, consistent with the fact that the current density was not modified. β3-subunit expression also altered the Nav1.7 band pattern in the biotinylated fractions. The upper band overlapped with the lower band under the migrating conditions used. The β3-subunit significantly increased (~7-fold) the intensity of the lower band as compared to the lower band of control, consistent with the increase of the Nav1.7 current density elicited by the β3-subunit (Figure 1C). Finally, β4-subunit co-transfection led to a small but significant decrease of the lower band.

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