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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.


The different forms of Nav1.7 observed with β-subunits are due to differential glycosylation patterns. Western blot of a biotinylation assay followed by deglycosylation with total lysate and cell surface fractions from HEK293 cells transiently transfected with Nav1.7 alone, or co-expressed with each individual β-subunit. Samples were non-treated or treated with Peptide: N-Glycosidase F (PNGaseF) to remove glycosylated residues of the protein. The total lysate Nav1.7 band (black/white triangle) was slightly shifted to an apparent lower molecular weight (gray triangle) when treated with PNGaseF. In the biotinylation fraction, the pattern of Nav1.7 glycosylation by the β-subunits was the same as in Figure 4.(black and white triangles). When treated with PNGaseF, all the different bands shifted to the lower band of the same apparent molecular weight, irrespective of the β-subunit co-expressed.
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Figure 6: The different forms of Nav1.7 observed with β-subunits are due to differential glycosylation patterns. Western blot of a biotinylation assay followed by deglycosylation with total lysate and cell surface fractions from HEK293 cells transiently transfected with Nav1.7 alone, or co-expressed with each individual β-subunit. Samples were non-treated or treated with Peptide: N-Glycosidase F (PNGaseF) to remove glycosylated residues of the protein. The total lysate Nav1.7 band (black/white triangle) was slightly shifted to an apparent lower molecular weight (gray triangle) when treated with PNGaseF. In the biotinylation fraction, the pattern of Nav1.7 glycosylation by the β-subunits was the same as in Figure 4.(black and white triangles). When treated with PNGaseF, all the different bands shifted to the lower band of the same apparent molecular weight, irrespective of the β-subunit co-expressed.

Mentions: To confirm that the different bands observed when β-subunits are coexpressed, particularly β1 and β3-subunits, represent alternative glycosylated form of Nav1.7, we again incubated the input and biotinylated fractions with PNGaseF. A small but consistent shift of the Nav1.7 band into a lower apparent molecular weight band in the total cell lysate fraction was observed (the white arrow heads shifted to the gray arrow heads, Figure 6). Furthermore, when incubating the biotinylated fraction of β-subunit and Nav1.7 co-expression experiments with PNGaseF, all of the Nav1.7 bands shifted to a single band (gray arrow heads) of the same molecular weight. This confirms that the β1- and β3-subunits modulate differential glycosylation patterns on Nav1.7 (Figure 6, black and white arrow heads). Noteworthy, when comparing the single band of Nav1.7 when samples are treated with PNGaseF in the input fraction with the one in the biotinylated fraction, it seems that this band migrates slower in the input as compared to biotinylated fraction when β-subunits are coexpressed (compare bands highlighted by gray arrows for each blots). This may be due to other post-translational modification such as sialylation or palmitoylation of the channel. Further experiments using desialylation or depalmitoylation treatment are needed to confirm this possibility.


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 different forms of Nav1.7 observed with β-subunits are due to differential glycosylation patterns. Western blot of a biotinylation assay followed by deglycosylation with total lysate and cell surface fractions from HEK293 cells transiently transfected with Nav1.7 alone, or co-expressed with each individual β-subunit. Samples were non-treated or treated with Peptide: N-Glycosidase F (PNGaseF) to remove glycosylated residues of the protein. The total lysate Nav1.7 band (black/white triangle) was slightly shifted to an apparent lower molecular weight (gray triangle) when treated with PNGaseF. In the biotinylation fraction, the pattern of Nav1.7 glycosylation by the β-subunits was the same as in Figure 4.(black and white triangles). When treated with PNGaseF, all the different bands shifted to the lower band of the same apparent molecular weight, irrespective of the β-subunit co-expressed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: The different forms of Nav1.7 observed with β-subunits are due to differential glycosylation patterns. Western blot of a biotinylation assay followed by deglycosylation with total lysate and cell surface fractions from HEK293 cells transiently transfected with Nav1.7 alone, or co-expressed with each individual β-subunit. Samples were non-treated or treated with Peptide: N-Glycosidase F (PNGaseF) to remove glycosylated residues of the protein. The total lysate Nav1.7 band (black/white triangle) was slightly shifted to an apparent lower molecular weight (gray triangle) when treated with PNGaseF. In the biotinylation fraction, the pattern of Nav1.7 glycosylation by the β-subunits was the same as in Figure 4.(black and white triangles). When treated with PNGaseF, all the different bands shifted to the lower band of the same apparent molecular weight, irrespective of the β-subunit co-expressed.
Mentions: To confirm that the different bands observed when β-subunits are coexpressed, particularly β1 and β3-subunits, represent alternative glycosylated form of Nav1.7, we again incubated the input and biotinylated fractions with PNGaseF. A small but consistent shift of the Nav1.7 band into a lower apparent molecular weight band in the total cell lysate fraction was observed (the white arrow heads shifted to the gray arrow heads, Figure 6). Furthermore, when incubating the biotinylated fraction of β-subunit and Nav1.7 co-expression experiments with PNGaseF, all of the Nav1.7 bands shifted to a single band (gray arrow heads) of the same molecular weight. This confirms that the β1- and β3-subunits modulate differential glycosylation patterns on Nav1.7 (Figure 6, black and white arrow heads). Noteworthy, when comparing the single band of Nav1.7 when samples are treated with PNGaseF in the input fraction with the one in the biotinylated fraction, it seems that this band migrates slower in the input as compared to biotinylated fraction when β-subunits are coexpressed (compare bands highlighted by gray arrows for each blots). This may be due to other post-translational modification such as sialylation or palmitoylation of the channel. Further experiments using desialylation or depalmitoylation treatment are needed to confirm this possibility.

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.