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Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons.

Bao L - Mol Pain (2015)

Bottom Line: Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2.Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex.These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, 200031, China. baolan@sibcb.ac.cn.

ABSTRACT
Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

No MeSH data available.


Related in: MedlinePlus

Model molecules to identify the signals in Navs that mediate the trafficking regulation. Navs consist of four domains (I, II, III and IV) connected by three intracellular loops (L1–L3); each domain is formed by six transmembrane segments (TM; S1–S6). Both the N-terminus (N) and the C-terminus (C) of Navs are located in the cytoplasm. CD8α and TFR1, which have distinct cell-surface localization, are adapted to detect the roles that particular regions of the Navs have in subcellular distribution. The type I membrane protein CD8α is suitable for testing three intracellular loops, the C-terminus and the transmembrane segments that pass through the membrane in the extracellular to intracellular direction (S2, S4 and S6), whereas the type II membrane protein TFR1 is appropriate for testing the N-terminus and the transmembrane segments that pass through the membrane in the opposite direction (S1, S3 and S5). A Myc tag is inserted to the N-terminus of CD8α or the C-terminus of TFR1, and non-permeabilized immunostaining is performed with Myc antibody in transfected living cells to label these proteins on the plasma membrane. A Flag tag is inserted to the N-terminus of TFR1 for the permeabilized immunostaining with Flag antibody to label the protein in whole cell. The sequence of CD8α or TFR1 is replaced with corresponding region of Nav, such as Myc-CD8α(TMIVS6), Myc-CD8α-C, Flag-TFR1(TMIVS1)-Myc and N-TFR1-Myc. This figure is adapted from Li et al. [15]
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Fig1: Model molecules to identify the signals in Navs that mediate the trafficking regulation. Navs consist of four domains (I, II, III and IV) connected by three intracellular loops (L1–L3); each domain is formed by six transmembrane segments (TM; S1–S6). Both the N-terminus (N) and the C-terminus (C) of Navs are located in the cytoplasm. CD8α and TFR1, which have distinct cell-surface localization, are adapted to detect the roles that particular regions of the Navs have in subcellular distribution. The type I membrane protein CD8α is suitable for testing three intracellular loops, the C-terminus and the transmembrane segments that pass through the membrane in the extracellular to intracellular direction (S2, S4 and S6), whereas the type II membrane protein TFR1 is appropriate for testing the N-terminus and the transmembrane segments that pass through the membrane in the opposite direction (S1, S3 and S5). A Myc tag is inserted to the N-terminus of CD8α or the C-terminus of TFR1, and non-permeabilized immunostaining is performed with Myc antibody in transfected living cells to label these proteins on the plasma membrane. A Flag tag is inserted to the N-terminus of TFR1 for the permeabilized immunostaining with Flag antibody to label the protein in whole cell. The sequence of CD8α or TFR1 is replaced with corresponding region of Nav, such as Myc-CD8α(TMIVS6), Myc-CD8α-C, Flag-TFR1(TMIVS1)-Myc and N-TFR1-Myc. This figure is adapted from Li et al. [15]

Mentions: Navs consist of four domains connected by three intracellular loops; each domain is formed by six transmembrane segments. Both the N-terminus and the C-terminus of Navs are located in the cytoplasm. To identify the amino acids, motifs or sequences in Navs that mediate the trafficking regulation, model molecules such as CD4, CD8α and transferring receptor 1 (TFR1), which have distinct cell surface localization, can be adapted to detect the roles that particular regions of the Navs have in subcellular distribution (Fig. 1) [11, 17, 18]. To ascertain the effect of intracellular and transmembrane segments, the orientation of the Nav intracellular sequence and transmembrane segment within the membrane should be considered when constructing the adapted molecule. The type I membrane protein CD8α is suitable for testing three intracellular loops, the C-terminus and the transmembrane segments that pass through the membrane in the extracellular to intracellular direction, whereas the type II membrane protein TFR1 is appropriate for testing the N-terminus and the transmembrane segments that pass through the membrane in the opposite direction (Fig. 1) [18]. Additionally, the specific cells used to examine distinct subcellular structures should be considered. For example, because neurons are round and their subcellular structures are occasionally obscured, COS-7 cells, which are derived from African green monkey kidney fibroblast-like cells and are flat, are usually used to analyze the subcellular localization of proteins in organelles. Importantly, the amino acids, motifs and sequences that are thought to mediate the regulation of Nav trafficking should ultimately be tested by point mutation or sequence replacement in full-length channels to evaluate their role in subcellular localization.Fig. 1


Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons.

Bao L - Mol Pain (2015)

Model molecules to identify the signals in Navs that mediate the trafficking regulation. Navs consist of four domains (I, II, III and IV) connected by three intracellular loops (L1–L3); each domain is formed by six transmembrane segments (TM; S1–S6). Both the N-terminus (N) and the C-terminus (C) of Navs are located in the cytoplasm. CD8α and TFR1, which have distinct cell-surface localization, are adapted to detect the roles that particular regions of the Navs have in subcellular distribution. The type I membrane protein CD8α is suitable for testing three intracellular loops, the C-terminus and the transmembrane segments that pass through the membrane in the extracellular to intracellular direction (S2, S4 and S6), whereas the type II membrane protein TFR1 is appropriate for testing the N-terminus and the transmembrane segments that pass through the membrane in the opposite direction (S1, S3 and S5). A Myc tag is inserted to the N-terminus of CD8α or the C-terminus of TFR1, and non-permeabilized immunostaining is performed with Myc antibody in transfected living cells to label these proteins on the plasma membrane. A Flag tag is inserted to the N-terminus of TFR1 for the permeabilized immunostaining with Flag antibody to label the protein in whole cell. The sequence of CD8α or TFR1 is replaced with corresponding region of Nav, such as Myc-CD8α(TMIVS6), Myc-CD8α-C, Flag-TFR1(TMIVS1)-Myc and N-TFR1-Myc. This figure is adapted from Li et al. [15]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4590712&req=5

Fig1: Model molecules to identify the signals in Navs that mediate the trafficking regulation. Navs consist of four domains (I, II, III and IV) connected by three intracellular loops (L1–L3); each domain is formed by six transmembrane segments (TM; S1–S6). Both the N-terminus (N) and the C-terminus (C) of Navs are located in the cytoplasm. CD8α and TFR1, which have distinct cell-surface localization, are adapted to detect the roles that particular regions of the Navs have in subcellular distribution. The type I membrane protein CD8α is suitable for testing three intracellular loops, the C-terminus and the transmembrane segments that pass through the membrane in the extracellular to intracellular direction (S2, S4 and S6), whereas the type II membrane protein TFR1 is appropriate for testing the N-terminus and the transmembrane segments that pass through the membrane in the opposite direction (S1, S3 and S5). A Myc tag is inserted to the N-terminus of CD8α or the C-terminus of TFR1, and non-permeabilized immunostaining is performed with Myc antibody in transfected living cells to label these proteins on the plasma membrane. A Flag tag is inserted to the N-terminus of TFR1 for the permeabilized immunostaining with Flag antibody to label the protein in whole cell. The sequence of CD8α or TFR1 is replaced with corresponding region of Nav, such as Myc-CD8α(TMIVS6), Myc-CD8α-C, Flag-TFR1(TMIVS1)-Myc and N-TFR1-Myc. This figure is adapted from Li et al. [15]
Mentions: Navs consist of four domains connected by three intracellular loops; each domain is formed by six transmembrane segments. Both the N-terminus and the C-terminus of Navs are located in the cytoplasm. To identify the amino acids, motifs or sequences in Navs that mediate the trafficking regulation, model molecules such as CD4, CD8α and transferring receptor 1 (TFR1), which have distinct cell surface localization, can be adapted to detect the roles that particular regions of the Navs have in subcellular distribution (Fig. 1) [11, 17, 18]. To ascertain the effect of intracellular and transmembrane segments, the orientation of the Nav intracellular sequence and transmembrane segment within the membrane should be considered when constructing the adapted molecule. The type I membrane protein CD8α is suitable for testing three intracellular loops, the C-terminus and the transmembrane segments that pass through the membrane in the extracellular to intracellular direction, whereas the type II membrane protein TFR1 is appropriate for testing the N-terminus and the transmembrane segments that pass through the membrane in the opposite direction (Fig. 1) [18]. Additionally, the specific cells used to examine distinct subcellular structures should be considered. For example, because neurons are round and their subcellular structures are occasionally obscured, COS-7 cells, which are derived from African green monkey kidney fibroblast-like cells and are flat, are usually used to analyze the subcellular localization of proteins in organelles. Importantly, the amino acids, motifs and sequences that are thought to mediate the regulation of Nav trafficking should ultimately be tested by point mutation or sequence replacement in full-length channels to evaluate their role in subcellular localization.Fig. 1

Bottom Line: Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2.Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex.These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, 200031, China. baolan@sibcb.ac.cn.

ABSTRACT
Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

No MeSH data available.


Related in: MedlinePlus