Sumoylation of Na V 1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

Abstract

The mechanism for the earliest response of central neurons to hypoxia-an increase in voltage-gated sodium current (I Na )-has been unknown. Here, we show that hypoxia activates the Small Ubiquitin-like Modifier (SUMO) pathway in rat cerebellar granule neurons (CGN) and that SUMOylation of Na V 1.2 channels increases I Na . The time-course for SUMOylation of single Na V 1.2 channels at the cell surface and changes in I Na coincide, and both are prevented by mutation of Na V 1.2-Lys38 or application of a deSUMOylating enzyme. Within 40 s, hypoxia-induced linkage of SUMO1 to the channels is complete, shifting the voltage-dependence of channel activation so that depolarizing steps evoke larger sodium currents. Given the recognized role of I Na in hypoxic brain damage, the SUMO pathway and Na V 1.2 are identified as potential targets for neuroprotective interventions.

Hypoxia Produces Pro-arrhythmic Late Sodium Current in Cardiac Myocytes by SUMOylation of NaV1.5 Channels

“…Tagging the N terminus of Na V 1.5 (mTFP-Na V 1.5) did not alter the biophysical properties of the channel nor its responsiveness to hypoxia or SUMO1 or SENP1 in the recording pipette (Figures S2; Table S1). Similarly, tagging the N terminus of SUMO1 (YFP-SUMO1) did not alter its operation with Na V 1.5, as previously observed with Na V 1.2 and three K + channels (Xiong et al, 2017;Plant et al, 2010Plant et al, , 2011Plant et al, , 2012Plant et al, , 2016.…”

“…Indicating FRET, and thus the intimate association of the two fluorophores consistent with SUMOylation, the time course of mTFP1 photobleaching under continuous illumination had a time constant (t) that was prolonged from less than 10 s to 27 s when the channel was expressed with YFP-SUMO1 ( Figures 2B and 2C). mTFP-Na V 1.5 also showed FRET with YFP-Ubc9, the SUMO conjugating enzyme that binds to target proteins only when a lysine subject to modification is present (Xiong et al, 2017;Plant et al, 2016). In contrast, FRET was not observed when mTFP-Na V 1.5 was expressed with linkage-incompetent YFP-SUMO1 95 (a variant that lacks the terminal Gly-Gly motif present in SUMO1 and the matured isoform SUMO1 97 ) or soluble YFP; in both cases, t was less than 10 s.…”

“…We delivered 1 nM purified SUMO1 peptide into iPS-CMs by the patch pipette; a concentration that evoked maximal effects on Na V 1.2 channels in cerebellar granule neurons (CGNs) and three different K + channels in CGNs, hippocampal neurons, and rat cardiac myocytes (Xiong et al, 2017;Plant et al, 2011Plant et al, , 2012Plant et al, , 2016. SUMO1 induced changes in cardiac I Na like those produced by hypoxia: I LATE increased to $4.2% of peak I Na without a change in the peak magnitude ( Figures 1B and 1C; Table 1).…”

“…SUMOylation is the enzyme-mediated linkage of one of three SUMO isoforms to the ε amino group of specific lysine residues in a target protein (Henley et al, 2014). Present in all eukaryotic cells, the SUMO pathway was known to regulate the trafficking and activity of nuclear transcription factors when we described it to operate as well at the plasma membrane by direct SUMOylation of Na + and K + channels to regulate excitability Plant et al, 2010Plant et al, , 2011Plant et al, , 2012Plant et al, , 2016Xiong et al, 2017). Given the important role of hypoxia-induced increases in I LATE in heart disease, we tested the hypothesis that SUMOylation of Na V 1.5 channels was the underlying mechanism.…”

Hydrogen sulfide inhibits Kir2 and Kir3 channels by decreasing sensitivity to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)

“…TIRF microscopy --Single protein particle complexes at the surface of live CHO cells were identified and studied by TIRF microscopy as previously described (40). Briefly, the critical angle for TIRF was adjusted using a CellTIRF illuminator (Olympus, Waltham, MA) and a high numerical-aperture apochromat objective (150x, 1.45 NA; Olympus) mounted on an automated fluorescence microscope (IX81; Olympus), controlled by Metamorph software (Molecular Devices).…”