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?1- and ?3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells.

ABSTRACT: Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming ?-subunit and associated ?-subunits that regulate Nav ?-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav ?-subunit also directly affects Navs gating. ?-subunits and glycosylation thus comodulate Nav ?-subunit gating. We hypothesized that ?-subunits could directly influence ?-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both ?1- and ?3-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 ?-subunits exist in multiple glycosylated states. The ?-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 ?1-subunits were coexpressed, suggesting that the ?1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the ?1-subunit increased the expression of this alternative glycosylated form and the ?3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for ?1- and ?3-subunits in the modulation of Nav1.7 ?-subunit glycosylation and cell surface expression.

SUBMITTER: Laedermann CJ

PROVIDER: S-EPMC3757325 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells.

Laedermann Cédric J CJ Syam Ninda N Pertin Marie M Decosterd Isabelle I Abriel Hugues H

Frontiers in cellular neuroscience 20130830

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 coexp ...[more]

PMID: 24009557

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Project description:Background:Voltage-gated sodium (NaV) channels are heteromeric proteins consisting of a single pore forming ?-subunit associated with one or two auxiliary ?-subunits. These channels are classically known for being responsible of action potential generation and propagation in excitable cells; but lately they have been reported as widely expressed and regulated in several human cancer types. We have previously demonstrated the overexpression of NaV1.6 channel in cervical cancer (CeCa) biopsies and primary cultures, and its contribution to cell migration and invasiveness. Here, we investigated the expression of NaV channels ?-subunits (NaV?s) in the CeCa cell lines HeLa, SiHa and CaSki, and determined their contribution to cell proliferation, migration and invasiveness. Methods:We assessed the expression of NaV?s in CeCa cell lines by performing RT-PCR and western blotting experiments. We also evaluated CeCa cell lines proliferation, migration, and invasion by in vitro assays, both in basal conditions and after inducing changes in NaV?s levels by transfecting specific cDNAs or siRNAs. The potential role of NaV?s in modulating the expression of NaV ?-subunits in the plasma membrane of CeCa cells was examined by the patch-clamp whole-cell technique. Furthermore, we investigated the role of NaV?1 on cell cycle in SiHa cells by flow cytometry. Results:We found that the four NaV?s are expressed in the three CeCa cell lines, even in the absence of functional NaV ?-subunit expression in the plasma membrane. Functional in vitro assays showed differential roles for NaV?1 and NaV?4, the latter as a cell invasiveness repressor and the former as a migration abolisher in CeCa cells. In silico analysis of NaV?4 expression in cervical tissues corroborated the downregulation of this protein expression in CeCa vs normal cervix, supporting the evidence of NaV?4's role as a cell invasiveness repressor. Conclusions:Our results contribute to the recent conception about NaV?s as multifunctional proteins involved in cell processes like ion channel regulation, cell adhesion and motility, and even in metastatic cell behaviors. These non-canonical functions of NaV?s are independent of the presence of functional NaV ?-subunits in the plasma membrane and might represent a new therapeutic target for the treatment of cervical cancer.

Dataset Information

?1- and ?3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells.

Publications

β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells.

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