his senp1 protein  (Boston Biochem)

Journal: iScience

Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

doi: 10.1016/j.isci.2022.104969

Figure Lengend Snippet: Acute hypoxia inhibits I K1 via SUMOylation I K1 in rat ventricular cardiomyocytes (RVCMs) was studied by the whole-cell patch-clamp. Currents were evoked in a high external K + recording buffer and measured at −120 mV, as described in the . Hypoxia was a drop in O 2 from ambient levels to 2% (red), measured at the cell. The time-course of hypoxic inhibition was studied by recording the magnitude of I K1 every second. Cells were studied with the following pipette solutions: control (blue), SUMO1 (1 μM, orange), or SENP1 (2 μM, magenta). CRY2-SENP1 was targeted to the cell membrane by the activation of a 460 nm LED during the time period indicated. I K1 currents were blocked by the addition of 3 mM Ba 2+ (green). (A) Exposure to acute hypoxia (2% O 2 ) inhibits ∼40% of I K1 in RVCMs. Left , representative current sweeps; right , a representative time course showing the kinetics of hypoxic inhibition in RVCMs studied with a control pipette solution. The inhibition is precluded by including SENP1 (2 μM) in the recording pipette. The reverse ramp recording protocol is inset. (B) Including SUMO1 (1 μM) in the recording pipette inhibits I K1 and precludes the effects of hypoxia. SENP1 augments I K1 and protects the current from hypoxia. Data are from 8 to 10 RVCMs per group, ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test. (C) Hypoxic inhibition of I K1 was reversed by the activation of CRY2-SENP1 with 460 nm light (cyan box) when RVCMs are studied with a control pipette solution. Right , CRY2-SENP1 does not alter current when 2 μM SENP1 is included in the recording pipette. Data are from 6 RVCMs, ∗p < 0 . 05 , ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test.

Article Snippet: Human His 6 -SENP1 , Boston Biochemical , Cat# E700.

Techniques: Patch Clamp, Inhibition, Transferring, Control, Membrane, Activation Assay, Two Tailed Test

Journal: iScience

Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

doi: 10.1016/j.isci.2022.104969

Figure Lengend Snippet: Lysine 49 is required for hypoxic inhibition of Kir2.1 Rat Kir2.1 channels were expressed in HEK293T cells and studied by whole-cell patch-clamp recording with the following pipette solutions: control (blue) SUMO1 (1 μM, orange) or SENP1 (2 μM, magenta). Currents were evoked using a high K + recording buffer and were measured at −80 mV, as described in the using a ramp protocol (inset). Hypoxia (red) was a drop in O 2 from ambient levels to 2%, measured at the cell. Kir2.1 channel currents are blocked by the addition of 3 mM Ba 2+ (green). The time course of hypoxic- inhibition was studied by recording the magnitude of I K1 every second. See also Figure S2 . (A) Representative sweeps showing hypoxic inhibition of Kir2.1 channels. (B) A representative time course showing that Kir2.1 is inhibited by hypoxia. (C) Including SUMO1 in the recording pipette decreases Kir2.1 currents and prevents further inhibition by hypoxia, while SENP1 augments the current and precludes the effects of hypoxia. Data are from 8 to 12 cells per group, ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test. (D) Kir2.1-K49Q channels are insensitive to regulation by hypoxia or inclusion of SUMO1 or SENP1 in the recording pipette. Data are from 8 to 12 cells per group.

Article Snippet: Human His 6 -SENP1 , Boston Biochemical , Cat# E700.

Techniques: Inhibition, Patch Clamp, Transferring, Control, Two Tailed Test

Journal: iScience

Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

doi: 10.1016/j.isci.2022.104969

Figure Lengend Snippet: SUMO1 decreases the effect of PIP 2 on Kir2.1 Rat Kir2.1 channel currents were studied in Xenopus oocytes by two-electrode voltage-clamp. Currents were evoked in a high external K + recording buffer and measured at −80 mV, as described in the . To study the effects of SUMOylation and deSUMOylation, the oocytes were also injected with cRNAs expressing Ubc9 and SUMO1 (orange) or SENP1 (magenta) and compared to control oocytes (blue). Endogenous PIP 2 was dephosphorylated at the 5′ position by co-expressing CIBN-CAAX and CRY2-5′ptase OCRL .(5′ptase) and activating this optogenetic system with a 460 nm LED were indicated by the cyan box. Data are mean ± s.d. for 6-8 oocytes per group, ∗p < 0 . 05 ,∗∗p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test; ∗p < 0 . 05 , unpaired, two-tailed Student’s t test. See also Figures S3–S6 . (A) Example time courses show that the rate of inhibition of Kir2.1 by the activation of CRY-5′ptase is increased by SUMO1 and slowed by SENP1. (B) The tau of inhibition is determined by fitting the data with a mono-exponential function. (C) Left , Summary data showing the effect of co-expressed SUMO1 or SENP1 on the peak Kir2.1 current before and after the activation of 5′ptase as well as the percentage of the current remaining ( middle ) and the tau of inhibition ( right ). (D) Left , Summary data showing that co-expression of SUMO1 or SENP1 does not alter peak Kir2.1-K49Q current before and after the activation of 5′ptase. The percentage of Kir2.1-K49Q current remaining ( middle ) and the tau of inhibition ( right ) are insensitive to SUMO1 or SENP1.

Article Snippet: Human His 6 -SENP1 , Boston Biochemical , Cat# E700.

Techniques: Injection, Expressing, Control, Two Tailed Test, Inhibition, Activation Assay

Journal: iScience

Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

doi: 10.1016/j.isci.2022.104969

Figure Lengend Snippet: PIP 2 opposed hypoxic inhibition of I K1 I K1 was studied in rat ventricular cardiomyocytes (RVCMs) using a whole-cell patch-clamp recording. To the knockdown Kir2.1 expression, RVCMs were transduced with lentiviral particles carrying eGFP and shRNA targeting KCNJ2. Kir2.1 knockdown cells (Kir2.1 kd -CMs) were identified by the expression of eGFP. Cells were studied with a control pipette solution (blue), or with pipette solutions containing purified SUMO1 (1 μM, orange) or SENP1 (2 μM, magenta). Where indicated, the control pipette solution contained diC8 PIP 2 . Paired patch-clamp data were analyzed by Students t -test; ∗∗∗, p < 0.01. The proximity ligation assay (PLA) for native Kir2.1-SUMO1 interactions was performed as described in the and analyzed using an unpaired Mann-Whitney rank test, ∗∗∗∗p < 0.001. See also Figures S7 and . (A) Left , Example sweeps show that I K1 is diminished in Kir2.1 kd -CMs and the remaining current is insensitive to acute hypoxia. Kir2.1 kd -CMs are identified by expression of GFP ( inset , scale bar = 10 μm). Right , summary data from 8 to 10 cells per group show that the regulation of I K1 by hypoxia, SUMO1, and SENP1 is lost in Kir2.1 kd -CMs. (B) The magnitude and the regulation of I K1 by hypoxia, SUMO1, and SENP1 are unaltered when cells are treated with a control, scrambled shRNA; 7-8 cells per group. (C) Left , Example traces to show that including diC8-PIP 2 in the pipette solution reduces the hypoxic inhibition of I K1 in RVCMs in a concentration-dependent manner. The arrow indicates the change in current magnitude between exposure to ambient O 2 and 2% O 2 in the same cell. Right , Concentration-response curve showing that including diC8-PIP 2 in the pipette opposes hypoxic inhibition of I K1 in control RVCMs and RVCMs expressing the scrambled shRNA. The hypoxic-response is diminished in Kir2.1 kd -CMs. Data are mean ± s.d. for 7-8 cells per condition. (D) DiC8-PIP 2 decreases hypoxic inhibition of Kir2.1 channels expressed in HEK293T cells (control) in a concentration-dependent manner using the experimental paradigm described in C, above. Current inhibition is diminished when SENP1 is included in the recording pipette and is not observed when Kir2.1-K49Q channels are studied. Data are mean ± s.d. for 6 cells per condition. (E) PLA shows that native Kir2.1 colocalizes with SUMO1 in RVCMs and that Kir2.1-SUMO interactions are increased by acute hypoxia. Kir2.2 and Kir2.3 do not associate with SUMO1. Representative data are shown with DAPI-labeled nuclei in red and PLA interactions in green for ease of visualization. Summary data are obtained from multiple experiments each with multiple fields of view containing 20-30 nuclei. The scale bar is 10 μm.

Article Snippet: Human His 6 -SENP1 , Boston Biochemical , Cat# E700.

Techniques: Inhibition, Patch Clamp, Knockdown, Expressing, Transduction, shRNA, Control, Transferring, Purification, Proximity Ligation Assay, MANN-WHITNEY, Concentration Assay, Labeling

Journal: iScience

Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

doi: 10.1016/j.isci.2022.104969

Figure Lengend Snippet:

Article Snippet: Human His 6 -SENP1 , Boston Biochemical , Cat# E700.

Techniques: Recombinant, Virus, shRNA, Control, In Situ, Software, Microscopy