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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.


Only β1-subunit significantly increases Nav1.7 recovery from inactivation (RFI). (A–D) RFI from HEK293 cells transfected with Nav1.7 alone or co-transfected with individual β-subunits. Smooth curves were fitted incorporating a geometric weight to arrive at the final curve (no equation) and the t½ was calculated by interpolation on the x-axis from a linear relation between the 2 points juxtaposing half recovery (y1 < 0.5 < y2, see Materials and Methods). Only when co-expressed with β1-subunit (n = 27, p < 0.01) was Nav1.7 RFI significantly faster as compared to Nav1.7 alone (n = 72). β2- (n = 34), β3- (n = 16), and β4-subunits (n = 19) did not significantly alter RFI when co-expressed with Nav1.7. Individual points are the mean ± s.e.m. of the normalized current at each time point. Non-parametric One-Way analysis of variance (Kruskal-Wallis test) with Dunn post-hoc tests to compare each subunit co-expressed with Nav1.7 vs. Nav1.7 alone.
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Figure 3: Only β1-subunit significantly increases Nav1.7 recovery from inactivation (RFI). (A–D) RFI from HEK293 cells transfected with Nav1.7 alone or co-transfected with individual β-subunits. Smooth curves were fitted incorporating a geometric weight to arrive at the final curve (no equation) and the t½ was calculated by interpolation on the x-axis from a linear relation between the 2 points juxtaposing half recovery (y1 < 0.5 < y2, see Materials and Methods). Only when co-expressed with β1-subunit (n = 27, p < 0.01) was Nav1.7 RFI significantly faster as compared to Nav1.7 alone (n = 72). β2- (n = 34), β3- (n = 16), and β4-subunits (n = 19) did not significantly alter RFI when co-expressed with Nav1.7. Individual points are the mean ± s.e.m. of the normalized current at each time point. Non-parametric One-Way analysis of variance (Kruskal-Wallis test) with Dunn post-hoc tests to compare each subunit co-expressed with Nav1.7 vs. Nav1.7 alone.

Mentions: The influence of the β-subunits on recovery from inactivation (RFI) was also tested. Because the RFI relationships could not always be fitted with the exponential functions to the same degree, an interpolation from a linear relation between the 2 points juxtaposing half recovery to obtain the half-time (t½) of RFI was used. Only the β1-subunit significantly hastened t½ of RFI to 6.19 vs. 7.55 ms for the control (Figures 3A–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)

Only β1-subunit significantly increases Nav1.7 recovery from inactivation (RFI). (A–D) RFI from HEK293 cells transfected with Nav1.7 alone or co-transfected with individual β-subunits. Smooth curves were fitted incorporating a geometric weight to arrive at the final curve (no equation) and the t½ was calculated by interpolation on the x-axis from a linear relation between the 2 points juxtaposing half recovery (y1 < 0.5 < y2, see Materials and Methods). Only when co-expressed with β1-subunit (n = 27, p < 0.01) was Nav1.7 RFI significantly faster as compared to Nav1.7 alone (n = 72). β2- (n = 34), β3- (n = 16), and β4-subunits (n = 19) did not significantly alter RFI when co-expressed with Nav1.7. Individual points are the mean ± s.e.m. of the normalized current at each time point. Non-parametric One-Way analysis of variance (Kruskal-Wallis test) with Dunn post-hoc tests to compare each subunit co-expressed with Nav1.7 vs. Nav1.7 alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Only β1-subunit significantly increases Nav1.7 recovery from inactivation (RFI). (A–D) RFI from HEK293 cells transfected with Nav1.7 alone or co-transfected with individual β-subunits. Smooth curves were fitted incorporating a geometric weight to arrive at the final curve (no equation) and the t½ was calculated by interpolation on the x-axis from a linear relation between the 2 points juxtaposing half recovery (y1 < 0.5 < y2, see Materials and Methods). Only when co-expressed with β1-subunit (n = 27, p < 0.01) was Nav1.7 RFI significantly faster as compared to Nav1.7 alone (n = 72). β2- (n = 34), β3- (n = 16), and β4-subunits (n = 19) did not significantly alter RFI when co-expressed with Nav1.7. Individual points are the mean ± s.e.m. of the normalized current at each time point. Non-parametric One-Way analysis of variance (Kruskal-Wallis test) with Dunn post-hoc tests to compare each subunit co-expressed with Nav1.7 vs. Nav1.7 alone.
Mentions: The influence of the β-subunits on recovery from inactivation (RFI) was also tested. Because the RFI relationships could not always be fitted with the exponential functions to the same degree, an interpolation from a linear relation between the 2 points juxtaposing half recovery to obtain the half-time (t½) of RFI was used. Only the β1-subunit significantly hastened t½ of RFI to 6.19 vs. 7.55 ms for the control (Figures 3A–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.