Table 2.

Co-localization of SUMO1 with Na_V1.2 in response to hypoxia. CFP-tagged Na_V1.2 or Na_V1.2-Lys38Gln subunits were expressed in CHO cells with mCherry-SUMO1 (m-SUMO1) and studied by TIRFM and whole-cell patch-clamp (Figures 7 and 8). The number of photobleaching steps observed for each fluorophore in each single fluorescent spot reports on the stoichiometry of the channel complex. Na_V1.2 channels are monomers and show no more than one bleaching step when tagged with CFP (Figure 7). 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. SUMO1 was not observed to co-localize with Na_V1.2-Lys38Gln 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 40 s and almost all were co-localized with Na_V1.2. Whole-cell, peak current-density, measured at −20 mV, increased by ~70% within 40 s of hypoxia and remained stable during 2 min of hypoxia and 20 min of recovery at ambient levels of O₂. Pulse protocols to determine the activation (Act) and steady-state inactivation (SSI) V_1/2 values (the voltage evoking half-maximal conductance) were obtained as described in the Materials and methods and the manuscript Table. Data are means ± S.E.M. for 5 to 8 cells per group; * indicates p<0.05 compared with cells studied in ambient O₂ for each channel type studied.

DOI: http://dx.doi.org/10.7554/eLife.20054.013

Subunits expressed	CFP-NaV1.2 + m-SUMO1							CFP-NaV1.2-Lys38Gln + m-SUMO1
Condition	Ambient O2	Hypoxia 40 s	Hypoxia 2 min	Recovery 5 min	Recovery 10 min	Recovery 20 min	SENP1	Ambient O2	Hypoxia 40 s	Recovery 5 min
Single particle stoichiometry SUMO1: NaV1.2	1: 1	ND	1: 1	1: 1	ND	ND	0: 1	0: 1	0: 1	ND
Free CFP-NaV1.2 pixels / µm2	340 ± 16	139 ± 8	137 ± 11	134 ± 7	131 ± 6	129 ± 8	290 ± 10	309 + 12	303 + 15	305 + 12
Free mSUMO1 pixels / µm2	4 ± 5	12 ± 2	11 ± 3	10 ± 7	8 ± 6	8 ± 5	3 + 2	5 + 2	4 + 2	4 + 1
Co-localized pixels / µm2	67 ± 6	268 ± 12	265 ± 12	260 ± 11	245 ± 14	239 ± 8	3 + 2	2 + 1	2 + 2	1 + 2
Total CFP-NaV1.2 pixels / µm2	407 ± 10	407 ± 12	402 ± 10	394 ± 15	376 ± 14	362 ± 10	293 ± 8	311 ± 12	305 ± 16	306 ± 14
Act V½ (mV)	−22 ± 1.2	−31 ± 1.7	−32 ± 2	−29 + 2	−35 ± 1.4	−33 + 2	−3.5 ± 1.8	−4 + 2	−3.7 + 2.5	−3.1 ± 1.2
SSI V½ (mV)	−61 ± 2	−70 ± 3	−69 ± 4	−71 + 3	−70 ± 2	−68 + 3	−44 ± 1.5	−51 + 1.5	−53 + 3	−48 ± 2
IPeak (pA/pF)	−120 ± 8	−198 ± 10	−199 ± 13	−200 ± 14	−201 ± 13	−189 ± 17	−33 ± 12	−39 ± 13	−37 ± 9	−39 ± 14