Table 2.
Co-localization of SUMO1 with Nav1.5 in Response to Hypoxia
| Subunits Expressed | mTFP-NaV1.5 + m-SUMO1 | mTFP-NaV1.5-K442Q + m-SUMO1 | ||||||
|---|---|---|---|---|---|---|---|---|
| Condition | Ambient O2 | Hypoxia 50 s | Hypoxia 100 s | Recovery 150 s | Ambient O2 | Hypoxia 50 s | Hypoxia 100 s | Recovery 150 s |
| Single particle stoichiometry SUMO1: NaV1.5 | 1: 1 | 1: 1 | 1: 1 | 1: 1 | 0: 1 | 0: 1 | 0: 1 | 0: 1 |
| Free mTFP-NaV1.5, pixels/μm2 | 305 ± 21 | 166 ± 18* | 37 ± 4** | 39 ± 5** | 328 ± 26 | 333 ± 29 | 326 ± 25 | 308 ± 19 |
| Free m-SUMO1, pixels/μm2 | 3 ± 1 | 4 ± 2 | 7 ± 2 | 5 ± 2 | 3 ± 1 | 4 ± 1 | 4 ± 2 | 6 ± 2 |
| Co-localized pixels/μm2 | 33 ± 3 | 179 ± 15** | 301 ± 13** | 302 ± 15** | 4 ± 1 | 4 ± 2 | 3 ± 1 | 4 ± 1 |
NaV1.5 or NaV1.5-K442Q subunits tagged with mTFP1 were expressed in CHO-K1 cells with mCherry-SUMO1 (m-SUMO1) and studied by TIRFM and whole-cell patch clamp (Figures 3 and 5). The number of photobleaching steps observed for each fluorophore in each single fluorescent spot reports on the stoichiometry of the channel complex. NaV1.5 channels are monomers and show no more than one bleaching step when tagged with mTFP1 (Figure 5). No more than one bleaching steps was observed for mCherry-tagged SUMO1 subunits (free or co-localized with the channel). A 1:1 stoichiometry is maintained when cells are exposed to hypoxia (1.5% O2). SUMO1 was not observed to co-localize with NaV1.5-K442Q channels. The surface density of subunits was quantified as the mean of four 100- by 100-pixel regions for 6–10 cells per group. Exposure to hypoxia increased the number of SUMO1 monomers observed at the cell surface within 100 s, and almost all were co-localized with NaV1.5. Data are mean ± SEM for 5 to 8 cells per group;
p < 0.05,
p < 0.01 compared with cells studied in ambient O2 for each channel type.