Molecular Pathology of Sodium Channel Beta-Subunit Variants

Figures

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  FIGURE 1. Mapping of cardiac disease-linked variants in β1 or β3 subunits onto a structure of hNaV1.4-β1 complex. (A) A structure of human muscle sodium channel and β1-subunit (hNaV1.4- β1 complex) (PDB: 6AGF) shown with the point mutations related to atrial fibrillation (AF) and Brugada syndrome (BrS) on either β1 or β3 subunits. (B) The alignment of human β1 and β3 subunits marked with cardiac disease-linked variants in this study.

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  FIGURE 2. AF-linked β1 variants show diverse effects on NaV channel activation and inactivation curves, when compared to WT β1. Groups of 3–5 cells are reported as mean ± SEM. Data are fit with a Boltzmann equation. (A) Left: Voltage dependence of channel activation (G-V) and steady-state inactivation (SSI) curves of NaV1.5 alone (opened circle) and with WT β1 co-expression (filled circle). Middle: Time dependent NaV channel recovery from inactivation of NaV1.5 alone and with WT β1 co-expression. The channel recovery is recorded as the ratio of channel available post 200 ms-conditioning pulse at −20 mV after varying recovery intervals of −120 mV. Right: Voltage dependence of steady-state fluorescence (F-V) curves of VSD-III LFS alone and with WT β1 co-expression. (B) The G-V and SSI curves (left), recovery from inactivation (middle) and VSD-III F-V curve (right) of NaV1.5 co-expressed with R85H β1 (▲), in comparison to WT β1 (dashed line). (C) The G-V and SSI curves (left), recovery from inactivation (middle) and VSD-III F-V curve (right) of NaV1.5 co-expressed with D153N β1 (♦), in comparison to WT β1 (dashed line). (D) The G-V and SSI curves (left), recovery from inactivation (middle)…

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  FIGURE 3. AF-linked β3 variants alter the NaV1.5 voltage dependence activation and VSD-III fluorescence emission curves relative to WT β3. Groups of 3-5 cells are reported as mean ± SEM. Data are fit with a Boltzmann equation. (A) Left: The G-V and SSI curves of NaV1.5 alone (opened circle) and with WT β3 co-expression (filled circle). Middle: Time dependent NaV channel recovery from inactivation of NaV1.5 alone and with WT β3 co-expression. Right: Voltage dependence of steady-state fluorescence (F-V) curves of VSD-III VCF alone and with WT β3 co-expression. (B) The G-V and SSI curves (left), recovery from inactivation (middle) and VSD-III F-V curve (right) of NaV1.5 co-expressed with R6K β3 (▲), in comparison to WT β3 (dashed line). (C) The G-V and SSI curves (left), recovery from inactivation (middle) and VSD-III F-V curve (right) of NaV1.5 co-expressed with L10P β3 (♦), in comparison to WT β3 (dashed line). (D) The G-V and SSI curves (left), recovery from inactivation (middle) and VSD-III F-V curve (right) of NaV1.5 co-expressed with M161T β3 (■), in comparison to WT β3 (dashed line). (E) A bar graph shows the VSD-III deactivation time constants exponential fitted to the fluorescence…

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  FIGURE 4. AF-related β1, but not β3 variants affect the NaV channel surface expression, as illustrated by immuno-fluorescence staining of NaV1.5 α- and β-subunits. 4 days after injection, Xenopus oocytes were fixed, stained and imaged with confocal microscope. Scale bars represent 5 µm. (A) Xenopus oocytes co-injected with NaV1.5 and WT, R85H, D153N, or T189M β1-cmycs were immuno-stained with mouse anti-PanNaV antibody and rabbit anti-myc antibody. The surface expression of NaV1.5 and β1 were labeled in green and red accordingly. The last row showed the combined images of NaV1.5 and β1 labeling. (B) Xenopus oocytes co-injected with NaV1.5 and WT, R6K, L10P or M161T β3s were immuno-stained with mouse anti-PanNaV antibody and rabbit anti-scn3b antibody. The surface expression of NaV1.5 and β3 were labeled in green and red accordingly. The last row showed the combined images of NaV1.5 and β3 labeling.

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  FIGURE 5. BrS-linked β1 variant alters the NaV channel steady-state inactivation, and VSD-III activation relative to WT β1. Groups of 3–5 cells are reported as mean ± SEM. Data are fit with a Boltzmann equation. (A) The G-V and SSI curves (left), recovery from inactivation (middle) and VSD-III F-V curve (right) of NaV1.5 co-expressed with E87Q β1 (), compared to WT β1 (dashed line). (B) The G-V and SSI curves (left), recovery from inactivation (middle) and VSD-III F-V curve (right) of NaV1.5 co-expressed with V110I β3 (), in comparison to WT β3 (dashed line).

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  FIGURE 6. BrS-linked β3 variant reduces the peak current amplitude and the cell membrane expression of NaV channel complex, while BrS-linked β1 variant does not. Current is recorded by cut-open voltage clamp with the same cell area. Groups of 3–5 cells are report as mean ± SEM. Immunofluorescence staining is imaged by confocal microscope 4 days after an RNA injection. (A) The top graph shows the peak current-voltage relationships of NaV1.5 co-expressed with WT β1 (black) and E87Q β1 (blue), illustrating the comparable peak current amplitude. The bottom graph shows the peak current-voltage relationships of NaV1.5 co-expressed with WT β3 (black) and V110I β3 (blue). (B) The expression of NaV1.5 α- and β3-subunits on the Xenopus oocytes membrane immunostained by specific antibodies for NaV1.5 (green) and β3 (red).