Figure 5. Biochemical verification of the β2 and β4 disulfide bond with 910Cys in Nav1.2.
Top rows indicate particular Nav channel and β-subunit constructs with which oocytes were injected. WT proteins are indicated as such whereas mutants are noted with a residue number; a superscript letter symbolizes in which partner of the complex pair the mutation is found. rβ2 was loaded in each case except in the two rightmost lanes, where rβ4 was used. Labels on left column indicate which antibody was used to immunoblot the associated slice. (a) Western blot run under non-reducing conditions reveals a fraction of rβ2 migrating with WT rNav1.2a but not after selective cysteine substitution. Crude lysate from injected oocytes was run on a protein gel and probed for both the channel and the myc-tag of the β-subunit. The top PanNav slice and bottom myc slice show the expression of the respective proteins in lysates from oocytes injected with the indicated constructs. Even though equal quantities of protein was loaded in each lane (10 μg), the C910S channel mutant expresses less bountifully than the WT channel and as a result shows a weaker signal. The WT β-subunit can form redox-sensitive multimers that migrate at an apparent mass similar to that of the Nav channel. Mutation of 55Cys prevents multimer formation and disappearance of the high molecular weight band. Open arrows identify bands representing Nav channel-bound β-subunit and aid in distinguishing them from the multimeric β-subunit. Substituting 910Cys within rNav1.2a with Ser causes a loss of the channel-bound β-subunit band. rβ4 also binds to WT rNav1.2a, as evidenced by a second band, and no longer interacts with the C910S mutant. (b) Addition of DTT prevents both binding of rβ2 to the channel and formation of β-subunit multimers. The absence of myc signal at the same apparent weight as rNav1.2a indicates that binding to the channel is sensitive to reduction. In all cases, α-tubulin was used as a loading control. (c) WT rNav1.2a co-immunoprecipitates with rβ2 and rβ4. The β-subunit was pulled down with an antibody directed against a C-terminal myc-tag and treated with DTT before loading onto the gel. The channel is present only when the WT channel is co-expressed with the WT β-subunit.
Figure 5—figure supplement 1. Biochemical assessment of rβ2 oligomer formation.
