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First published online 9 September 2008
doi: 10.1242/jcs.026856

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The voltage-gated Na⁺ channel Na_v1.8 contains an ER-retention/retrieval signal antagonized by the β3 subunit

Zhen-Ning Zhang^*, Qian Li^*, Chao Liu, Hai-Bo Wang, Qiong Wang and Lan Bao

Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China

View larger version (69K): [in this window] [in a new window]   Fig. 1. The RRR motif in the first intracellular loop of Nav1.8 serves as an ER-retention/retrieval signal. (A) COS-7 cells transfected with Nav1.8-Myc were immunofluorescently labeled with antibodies against Myc and the ER marker calnexin, or the Golgi marker GM130. (B) Schematic of the intracellular segments of Nav1.8 and the amino acid sequence of the first part of the first intracellular loop (1L). (C) Plasmids expressing Myc-CD8, Myc-CD8-KKTN, or a series of constructs with the C-terminus of CD8 linked to the second intracellular loop (2L) or the various segments of 1L of Nav1.8 were transfected into COS-7 cells. Transfected cells were immunofluorescently labeled with antibody against Myc or calnexin. The images are representative of at least three independent experiments. Scale bars: 10 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 2. The RRR motif retains the protein in the ER. The maturation state of the indicated proteins in cell lysates of transfected COS-7 cells was examined by Endo H digestion. Representative immunoblots (IB) for the Myc tag show the mature (black arrowheads) and immature (open arrowheads) forms of the proteins. The experiments were repeated at least three times.

View larger version (71K): [in this window] [in a new window]   Fig. 3. The RRR ER-retention/retrieval signal reduces surface expression of the CD8 chimeras. COS-7 cells were transfected with plasmids expressing Myc-CD8, Myc-CD8-KKTN, or with a series of constructs with the C-terminus of CD8 linked to 2L or to the various segments of 1L of Nav1.8. Non-permeabilized and permeabilized immunofluorescence labeling were carried out with antibodies against Myc. The images are representative of at least three independent experiments. Scale bar: 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 4. The RRR ER-retention/retrieval signal reduces surface expression of Nav1.8. (A,B) COS-7 cells transfected with a plasmid expressing Myc-CD8, Myc-CD8-KKTN, or with a series of constructs with the C-terminus of CD8 linked to 2L or to the various segments of 1L of Nav1.8, were subjected to cell-surface biotinylation/immunoblotting. Representative immunoblot and quantitative analyses are shown. Actin served as an internal control for protein loading. The ratio of immunoblot intensity of surface versus total protein was calculated and data were plotted as a percentage of the controls (n3). **P<0.01 versus cells transfected with the plasmids indicated; ##P<0.01 versus Myc-CD8-1L-1-1-expressing cells. (C) HEK293 cells were transfected with plasmids expressing Nav1.8 or Nav1.8 mutants in which 495RRR497 was mutated to alanine (Nav1.8m-Myc), or the first intracellular loop was replaced by the second one (Nav1.8r-Myc), and subjected to cell-surface biotinylation/immunoblotting. Data were plotted as a percentage of Nav1.8-Myc. *P<0.05 versus cells transfected with Nav1.8-Myc (n=3).

View larger version (57K): [in this window] [in a new window]   Fig. 5. The C-terminus of the β3 subunit masks the RRR ER-retention/retrieval signal. (A) COS-7 cells were co-transfected with plasmids expressing Myc-CD8-1L-1-3 and β1-GFP or β3-GFP, and subjected to cell-surface biotinylation/immunoblotting. Representative immunoblot and quantitative analyses are shown. Actin served as an internal control for protein loading. Data were plotted as a percentage of controls. *P<0.05 versus cells transfected with Myc-CD8-1L-1-3 only (n=3). (B) COS-7 cells were co-transfected with plasmids expressing CD8β-GFP and Myc-CD8, CD8β-β3C-GFP and Myc-CD8-1L-1, CD8β-GFP and Myc-CD8-1L-1, or CD8β-β3C-GFP and Myc-CD8-KKTN. Cells were immunofluorescently labeled with antibody against Myc. The images are representative of at least three independent experiments. Scale bar: 10 µm. (C,D) COS-7 cells were co-transfected with CD8β-GFP or CD8β-β3C-GFP plus Myc-CD8-1L-1, Myc-CD8-1L-1-1, Myc-CD8-1L-1-2 or Myc-CD8-1L-1-3, and subjected to cell-surface biotinylation/immunoblotting. *P<0.05 versus cells co-transfected with CD8β-GFP (n=3).

View larger version (43K): [in this window] [in a new window]   Fig. 6. The C-terminus of the β3 subunit interacts with segments that contain the RRR motif and promotes their surface expression. The C-terminus of the β3 subunit with the transmembrane domain (TM+β3C-GFP) was co-transfected with Myc-CD8-1L-1, Myc-CD8-1L-1-2, Myc-CD8-1L-1-3 or Myc-CD8-1L-1-3m in COS-7 cells. (A,C) Proteins were immunoprecipitated with GFP-specific antibody and cell lysates subjected to immunoblotting with antibodies against Myc or GFP as indicated. All experiments were repeated three times. (B,D-F) Transfected cells were subjected to cell-surface biotinylation/immunoblotting. Representative immunoblots are shown. Actin served as an internal control for protein loading. Quantitative data were plotted as a percentage of controls. *P<0.05 versus the control cells or the cells indicated (n=3).

View larger version (41K): [in this window] [in a new window]   Fig. 7. Mutation of the RRR motif in Nav1.8 disrupts β3-subunit-promoted surface expression of the channel. (A) TM+β3C-GFP was co-expressed with either Nav1.8-Myc or Nav1.8m-Myc in HEK293 cells. Proteins were immunoprecipitated with Myc antibody and cell lysates subjected to immunoblotting with antibodies against GFP or Myc as indicated. The experiment was repeated three times. (B-D) Nav1.8-Myc was co-transfected with β3-GFP or TM+β3C-GFP (B), and Nav1.8m-Myc was co-transfected with TM+β3C-GFP (C) in HEK293 cells. Cultured DRG neurons were incubated with GST-Tat or GST-Tat-β3C (D). Cells were subjected to cell-surface biotinylation/immunoblotting. Representative immunoblot and quantitative analyses are shown. Actin served as an internal control for protein loading. Data were plotted as a percentage of controls. *P<0.05 versus cells transfected with Nav1.8-Myc or Nav1.8m-Myc, or treated with GST-Tat (n=3).

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