Figure 4. Hypoxia Recruits One SUMO1 Monomer to Each Cell Surface Na_V1.5 Channel.

Single mTFP-Na_V1.5 channels (blue) and SUMO1 tagged with mCherry (m-SUMO1, red) were studied in CHO-K1 cells by TIRFM. Stoichiometric (photobleaching) and pixel-by-pixel analysis for subunit density and co-localization were performed as described in the STAR Methods. Manders’ coefficients were assessed post hoc for 3–5 regions per cell, and co-localization was defined as the presence of both fluorophores at more than 30% of the maximum fluorescence level recorded in that stack (and their overlap is represented in the images as white pixels). The time course of hypoxic modulation of Na_V1.5 I_LATE was studied with steps from −100 mV to −30 mV every 10 s and normalized to the peak current. Data represent 5–8 cells, and biophysical parameters and single particle statistical analyses are summarized in Tables S1 and S3.

(A) Left: single co-localized particles with mCherry and TFP fluorescence were observed at the surface of cells expressing Na_V1.5 and SUMO1. The time courses for simultaneous photobleaching of the fluorophores revealed that complexes have one subunit of each type.

(B) Histogram of photobleaching steps showing that hypoxia increased single m-SUMO1 (red) subunits at the cell surface co-localized with mTFP-Na_V1.5 channels (blue), without a change in subunit stoichiometry.

(C) Left: the images show that in ambient O₂, the surface density of m-SUMO1 (top) is low compared to Na_V1.5 (middle) with little co-localization (bottom). Right: hypoxia recruits m-SUMO1 to the cell surface within ~100 s (top), where it is co-localized with Na_V1.5 channels (bottom); surface levels of Na_V1.5 were not observed to change (middle). The scale bar represents 10 μm.

(D) Histogram of surface density summarizing six cells studied as described in (A). In ambient O₂, the density of pixels per μm² with SUMO1 alone (red) was 3 ± 1, and 305 ± 21 for Na_V1.5 (blue). The density of pixels with both fluorophores (green) was 33 ± 3 per μm². Hypoxia increased co-localization to 301 ± 13 pixels per μm² and decreased the density of free Na_V1.5 channels (37 ± 4) without altering the density of free SUMO1 (6 ± 2).

(E) The time course for the hypoxia-induced increase in the co-localization of mTFP-Na_V1.5 and m-SUMO1 (Manders’ coefficient, black circle) and the magnitude of I_LATE, as a percentage of the peak current (open circle) were coincident. A mean Manders’ coefficient of 0.10 ± 0.01 measured in ambient O₂ increased to 0.91 ± 0.03 in ~100 s of exposure to hypoxia. The mean I_LATE rose from 0.46% ± 0.1% to 4.4% ± 0.8%. Increases in the Manders’ coefficient and late current versus peak current ratio were unchanged for at least 3 min after cells were restored to ambient O₂.