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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.


Proposed scheme of the different intracellular pathways of α-subunits depending on the presence of different β-subunits. After synthesis in the rough endoplasmic reticulum (RER), α-subunits are rapidly folded and undergo a first step of glycosylation in the smooth endoplasmic reticulum (SER). It is known that N-acetlyglucosamine and oligosaccharide chains are bound on Asp residues of the protein, a process known as core-glycosylation. Newly synthesized glycoproteins are then translocated into the Golgi network, where they are subject to a second step of more complex glycosylation, involving many different enzymes. Once matured, proteins eventually translocate to the plasma membrane. The present findings suggest that core-glycosylated proteins can also be found anchored at the membrane. By some yet undefined mechanism, the β1-subunit interferes with the second glycosylation step (***full-glycosylation on the scheme) which leads to a modification of the glycosylation pattern of the α-subunits. The β1-subunit enhances the core-glycosylated form of Nav1.7. This suggests that the β1- and β3-subunits already interact with the α-subunits before the step of full-glycosylation of the channel occurring in the Golgi network. By enhancing the differential glycosylation pattern of Nav1.7, it can be proposed that the β1- and β3-subunits promote stabilization of the channel at the plasma membrane. On the contrary, it is likely that the β2- and β4-subunits, which have no effect on the glycosylation nor the anchoring, only briefly interact with the α-subunits before translocation of the channel to the plasma membrane. For sake of simplicity, the shown glycosylation patterns are arbitrary. Nav1.7 is depicted as being “freely” expressed in ER/Golgi and membrane networks for easier interpretation of the scheme. However, Nav1.7 is embedded in the membranes of the different organelles.
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Figure 7: Proposed scheme of the different intracellular pathways of α-subunits depending on the presence of different β-subunits. After synthesis in the rough endoplasmic reticulum (RER), α-subunits are rapidly folded and undergo a first step of glycosylation in the smooth endoplasmic reticulum (SER). It is known that N-acetlyglucosamine and oligosaccharide chains are bound on Asp residues of the protein, a process known as core-glycosylation. Newly synthesized glycoproteins are then translocated into the Golgi network, where they are subject to a second step of more complex glycosylation, involving many different enzymes. Once matured, proteins eventually translocate to the plasma membrane. The present findings suggest that core-glycosylated proteins can also be found anchored at the membrane. By some yet undefined mechanism, the β1-subunit interferes with the second glycosylation step (***full-glycosylation on the scheme) which leads to a modification of the glycosylation pattern of the α-subunits. The β1-subunit enhances the core-glycosylated form of Nav1.7. This suggests that the β1- and β3-subunits already interact with the α-subunits before the step of full-glycosylation of the channel occurring in the Golgi network. By enhancing the differential glycosylation pattern of Nav1.7, it can be proposed that the β1- and β3-subunits promote stabilization of the channel at the plasma membrane. On the contrary, it is likely that the β2- and β4-subunits, which have no effect on the glycosylation nor the anchoring, only briefly interact with the α-subunits before translocation of the channel to the plasma membrane. For sake of simplicity, the shown glycosylation patterns are arbitrary. Nav1.7 is depicted as being “freely” expressed in ER/Golgi and membrane networks for easier interpretation of the scheme. However, Nav1.7 is embedded in the membranes of the different organelles.

Mentions: General kinetic models of biosynthesis (Schmidt and Catterall, 1987) have proposed that the β2-subunit interacts with the α-subunit right before anchoring at the membrane. The present findings are consistent with such a model since both β2- and β4-subunits did not influence Nav1.7 glycosylation, and most likely interacted after the Golgi network (Figure 7). The α−β1 complex was previously shown to associate in the ER, enhancing the trafficking to the plasma membrane (Zimmer et al., 2002). Another study demonstrated that β1- and β3-subunits increase the efficiency of channel trafficking from the ER to the plasma membrane (Fahmi et al., 2001). The β3-subunit has also been shown to mask the ER-retention signal (Zhang et al., 2008). The results of the present work confirm earlier demonstrated interactions between the β- and α-subunits, and suggest an additional function of both: β1- and β3-subunits interact with the α-subunit in the ER/Golgi, where they regulate the differential glycosylation of Nav α-subunits, which in turn modulates the stabilization of the channel at the cell membrane (Figure 7). The mechanisms underlying their interaction and mediation of α-subunit glycosylation, as well as the identification of other potential isoforms that would be subject to such regulation, remain to be investigated.


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)

Proposed scheme of the different intracellular pathways of α-subunits depending on the presence of different β-subunits. After synthesis in the rough endoplasmic reticulum (RER), α-subunits are rapidly folded and undergo a first step of glycosylation in the smooth endoplasmic reticulum (SER). It is known that N-acetlyglucosamine and oligosaccharide chains are bound on Asp residues of the protein, a process known as core-glycosylation. Newly synthesized glycoproteins are then translocated into the Golgi network, where they are subject to a second step of more complex glycosylation, involving many different enzymes. Once matured, proteins eventually translocate to the plasma membrane. The present findings suggest that core-glycosylated proteins can also be found anchored at the membrane. By some yet undefined mechanism, the β1-subunit interferes with the second glycosylation step (***full-glycosylation on the scheme) which leads to a modification of the glycosylation pattern of the α-subunits. The β1-subunit enhances the core-glycosylated form of Nav1.7. This suggests that the β1- and β3-subunits already interact with the α-subunits before the step of full-glycosylation of the channel occurring in the Golgi network. By enhancing the differential glycosylation pattern of Nav1.7, it can be proposed that the β1- and β3-subunits promote stabilization of the channel at the plasma membrane. On the contrary, it is likely that the β2- and β4-subunits, which have no effect on the glycosylation nor the anchoring, only briefly interact with the α-subunits before translocation of the channel to the plasma membrane. For sake of simplicity, the shown glycosylation patterns are arbitrary. Nav1.7 is depicted as being “freely” expressed in ER/Golgi and membrane networks for easier interpretation of the scheme. However, Nav1.7 is embedded in the membranes of the different organelles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Proposed scheme of the different intracellular pathways of α-subunits depending on the presence of different β-subunits. After synthesis in the rough endoplasmic reticulum (RER), α-subunits are rapidly folded and undergo a first step of glycosylation in the smooth endoplasmic reticulum (SER). It is known that N-acetlyglucosamine and oligosaccharide chains are bound on Asp residues of the protein, a process known as core-glycosylation. Newly synthesized glycoproteins are then translocated into the Golgi network, where they are subject to a second step of more complex glycosylation, involving many different enzymes. Once matured, proteins eventually translocate to the plasma membrane. The present findings suggest that core-glycosylated proteins can also be found anchored at the membrane. By some yet undefined mechanism, the β1-subunit interferes with the second glycosylation step (***full-glycosylation on the scheme) which leads to a modification of the glycosylation pattern of the α-subunits. The β1-subunit enhances the core-glycosylated form of Nav1.7. This suggests that the β1- and β3-subunits already interact with the α-subunits before the step of full-glycosylation of the channel occurring in the Golgi network. By enhancing the differential glycosylation pattern of Nav1.7, it can be proposed that the β1- and β3-subunits promote stabilization of the channel at the plasma membrane. On the contrary, it is likely that the β2- and β4-subunits, which have no effect on the glycosylation nor the anchoring, only briefly interact with the α-subunits before translocation of the channel to the plasma membrane. For sake of simplicity, the shown glycosylation patterns are arbitrary. Nav1.7 is depicted as being “freely” expressed in ER/Golgi and membrane networks for easier interpretation of the scheme. However, Nav1.7 is embedded in the membranes of the different organelles.
Mentions: General kinetic models of biosynthesis (Schmidt and Catterall, 1987) have proposed that the β2-subunit interacts with the α-subunit right before anchoring at the membrane. The present findings are consistent with such a model since both β2- and β4-subunits did not influence Nav1.7 glycosylation, and most likely interacted after the Golgi network (Figure 7). The α−β1 complex was previously shown to associate in the ER, enhancing the trafficking to the plasma membrane (Zimmer et al., 2002). Another study demonstrated that β1- and β3-subunits increase the efficiency of channel trafficking from the ER to the plasma membrane (Fahmi et al., 2001). The β3-subunit has also been shown to mask the ER-retention signal (Zhang et al., 2008). The results of the present work confirm earlier demonstrated interactions between the β- and α-subunits, and suggest an additional function of both: β1- and β3-subunits interact with the α-subunit in the ER/Golgi, where they regulate the differential glycosylation of Nav α-subunits, which in turn modulates the stabilization of the channel at the cell membrane (Figure 7). The mechanisms underlying their interaction and mediation of α-subunit glycosylation, as well as the identification of other potential isoforms that would be subject to such regulation, remain to be investigated.

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.