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Results: 6

1.
FIGURE 4.

FIGURE 4. From: Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel ? and ?2 Subunits.

Representative Na+ current traces. Representative Na+ currents evoked by a depolarizing pulse to 0 mV were obtained from HEKhNav1.1 cells transfected with β2WT-GFP, Φ-GFP, or β2C26A-GFP, as indicated.

Chunling Chen, et al. J Biol Chem. 2012 November 9;287(46):39061-39069.
2.
FIGURE 1.

FIGURE 1. From: Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel ? and ?2 Subunits.

Structural predictions for β2. A, alignment of the N-terminal regions of human β2, β4, and β1 with conserved cysteine residues highlighted in boldface type. Residue numbering corresponds to β2. B, left, the crystal structure of the Ig loop of human myelin P0 (30) was used as a template to show the predicted position of β2Cys-26 (cyan), β2C21 (magenta), and β2C98 (magenta). Right, the crystal structure of the Ig loop of human myelin P0 (30) was used as a template to show the predicted intramolecular disulfide bond between β2Cys-21 and β2Cys-98.

Chunling Chen, et al. J Biol Chem. 2012 November 9;287(46):39061-39069.
3.
FIGURE 6.

FIGURE 6. From: Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel ? and ?2 Subunits.

Map of conserved extracellular Cys residues within Na+ channel α subunits. Locations of cysteine residues within the topology of the α subunit are denoted by C. All extracellular cysteine residues map to S5-S6 loop regions. 12 candidate residues are extracellular, whereas two residues are embedded in the membrane as part of a P loop region and are indicated in black. The color of the letter C denotes the amount of evolutionary conservation. Conservation in all sequences tested is denoted by blue; conservation in mammalian and E. electricus Nav is denoted by green; and conservation in mammalian channels only is denoted by red.

Chunling Chen, et al. J Biol Chem. 2012 November 9;287(46):39061-39069.
4.
FIGURE 3.

FIGURE 3. From: Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel ? and ?2 Subunits.

β2WT, Φ, and β2C26A subunits are expressed at the cell surface. A, cell surface biotinylation was performed on HEKhNav1.1 cells transfected with β2WT, Φ, or β2C26A as described under “Experimental Procedures.” Immunoreactive bands were detected with anti-V5 antibody. The position of β2 subunit migration on the gel is indicated by the arrow. Similar to previous experiments to assess β1 subunit cell surface expression using this method (27), we observed multiple V5 immunoreactive bands, probably representing various levels of avidin attachment to β2. B, Na/K ATPase β1 subunit immunoreactivity of samples from A as a loading control for cell surface proteins (arrows). C, HEKhNav1.1 cells expressing β2WT-GFP (top), Φ-GFP (center), or β2C26A-GFP (bottom) were processed for immunocytochemistry as described under “Experimental Procedures.” Cells were visualized for GFP epifluorescence (green) or with an anti-GFP antibody (red; Alexa Fluor 568) by confocal microscopy. DAPI (blue) indicates cell nuclei. Scale bar, 10 μm. Results presented in this figure are representative of five independent experiments.

Chunling Chen, et al. J Biol Chem. 2012 November 9;287(46):39061-39069.
5.
FIGURE 5.

FIGURE 5. From: Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel ? and ?2 Subunits.

Covalent α-β2 linkage is critical for targeting of β2 to nodes of Ranvier and the AIS. A, targeting of β2 constructs to nodes of Ranvier. DRG neurons were nucleofected with WT or mutant β2-GFP constructs, as indicted, and then co-cultured with Schwann cells under myelinating conditions for ∼2 weeks. Cultures were fixed and analyzed by immunofluorescence staining. β2WT-GFP (green) accumulated at nodes and heminodes (arrows), whereas the mutant constructs failed to accumulate at these sites. Paranodes were stained with Caspr (red), and myelin segments were stained with MBP (blue). Scale bar, 5 μm. B, targeting of β2 constructs in hippocampal neurons. β2-GFP constructs were nucleofected into hippocampal neurons and analyzed at 18 days in vitro. β2WT-GFP (green) was enriched in the AIS, labeled by ankyrin G staining (red) (arrows). In contrast, Φ-GFP and β2C26A-GFP mutants (green) were equally distributed in axons and dendrites or preferentially concentrated in dendrites and were not enriched in the AIS. Dendrites were stained with MAP2 (blue). Scale bar, 10 μm. C, Triton X-100 extracts mutant β2 constructs from hippocampal neuron cultures. β2WT-GFP nucleofected hippocampal neuron cultures were extracted with Triton X-100 prior to fixation and then fixed and stained. The β2WT-GFP construct was retained at the AIS despite detergent treatment and was extracted from other sites. Both Φ-GFP and β2C26A-GFP were largely extracted from the neurons, including from the AIS, by detergent treatment. Arrows in the GFP staining panels delineate the positions of AIS; neuronal somata are located on the left. Scale bar, 5 μm.

Chunling Chen, et al. J Biol Chem. 2012 November 9;287(46):39061-39069.
6.
FIGURE 2.

FIGURE 2. From: Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel ? and ?2 Subunits.

Na+ channel β2 and α subunits are disulfide-linked at β2 residue Cys-26. Coimmunoprecipitation was performed with HEKhNav1.1 cells transiently transfected with V5-tagged β2WT or various β2 cysteine-to-alanine mutants, as indicated. Solubilized Nav1.1 complexes were immunoprecipitated with anti-pan-Na+ channel antibody and separated on non-reducing SDS-polyacrylamide gels as described under “Experimental Procedures.” Western blots were detected with anti-V5 antibody to visualize high α-β2 protein complexes (arrows). A, disulfide-linked α-β2WT complexes are detected, whereas α-φ and α-β2C26A are not detectable, indicating that disulfide linkage between subunits was lost. B, α-β2WT (lane 1), α-β2C43A (lane 2), and α-β2C43A/C46A/C143A (lane 7) complexes are detected. In contrast, α-β2C26A/C43A (lane 4) and α-β2C26A/C43A/C46A/C143A (lane 5) complexes are not detectable, indicating that disulfide linkage between subunits was lost. Lanes 3 and 6 were left empty. Results of similar experiments for the remaining β2 constructs are summarized in Table 1. C, Western blot analysis shows robust expression of β2WT and mutant constructs. Top, HEKhNav1.1 cells were transfected with V5-tagged β2WT, Φ, or β2C26A or with GFP alone, as indicated. Cells were then solubilized and analyzed by Western blot with anti-V5 antibody to confirm subunit expression. Bottom, the blot was stripped and reprobed with anti-α-tubulin as a loading control. Results presented in this figure are representative of five independent experiments.

Chunling Chen, et al. J Biol Chem. 2012 November 9;287(46):39061-39069.

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