Journal: bioRxiv
Article Title: Hypoxia Exacerbates Kir2.1 Channel Dysfunction in an Andersen-Tawil Syndrome Variant Through a SUMO-Dependent Mechanism
doi: 10.1101/2025.04.25.650734
Figure Lengend Snippet: FRET data aer donor (mTFP) photobleaching curves, fit with a mono-exponential function to yield a fluorescence decay time constant (tau). Mean taus represent 80-120 regions of interest per group across at least 3 biological replicates. Patch-clamp data are from 10-12 cells per group. Kir2.1 currents were measured at -80 mV. Hypoxia is a drop in O 2 from ambient levels to 2% measured at the cell. 2-D08 was used at 30 µM and was applied 3 hours before the experiment. Statistical differences were determined using a paired, two-tailed Student’s t test where *** is p< 0.001 and **** is P<0.0001. A, Left , Representative time-courses showing hypoxic-inhibition of Kir2.1 current (blue). The residual current is blocked by 3 mM Ba 2+ . Pretreatment with 2-D08 increases basal Kir2.1 current and precludes the effects of hypoxia (purple). Right , Summary histogram showing hypoxic inhibition of Kir2.1 for control and B, 2- D08 treated cells. C, Left, Example FRET photobleaching curves showing the decrease in mTFP1-Kir2.1 fluorescence in HEK293T cells in the presence of free YFP (green) or YFP-tagged Ubc9 (blue). Right, Summary histogram of photobleaching tau’s showing FRET between mTFP-tagged Kir2.1 and YFP-Ubc9, or the catalytically inert variant YFP-Ubc9-C93S is not prevented by 2-D08. In contrast, FRET between the channel and YFP-tagged SUMO1 or SUMO2 is prevented by pretreatment with 2-D08. D, Summary histogram of photobleaching tau’s showing FRET between mTFP-tagged Kir2.1-K49Q and Ubc9, or the catalytically inert variant Ubc9-C93S is not prevented by 2-D08. In contrast, FRET is not observed between mTFP-tagged Kir2.1-K49Q and YFP- tagged SUMO1 or SUMO2.
Article Snippet: TAK-981 was from MedChemExpress, 2-D08 was from Selleck Chem, and N106 was from Tocris.
Techniques: Fluorescence, Patch Clamp, Two Tailed Test, Inhibition, Control, Variant Assay
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