Table 2.

Co-localization of SUMO1 with Na_v1.5 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

Na_V1.5 or Na_V1.5-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. Na_V1.5 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 Na_V1.5-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 Na_V1.5. 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.