pka inhibitor kt5720  (Hello Bio Inc)

Journal: iScience

Article Title: Photoactivated adenylyl cyclase in cortical astrocytes promotes synaptic potentiation and reveals alterations in Huntington’s disease

doi: 10.1016/j.isci.2025.113640

Figure Lengend Snippet: Light activated DdPAC-induced synaptic potentiation (DdPAC-LTP) shares properties of theta burst-induced potentiation (TBS-LTP) (A) Theta-burst stimulation induced LTP in cortical slices. Left panel shows a schematic representation of the experimental setup detailing TBS in layer 4 barrel cortex and recording of evoked field potentials in layer 3/4. Middle panel shows the induction of TBS-LTP in the presence of PKA inhibitor (KT5720), NMDA receptor blockers (5,7 DCK, MK801, and AP5). Right panel summarizes the fEPSP slope effects at 60 min in the different conditions. (B) DdPAC stimulation by 660 nm light increases the slope of evoked fEPSPs. Left panel shows a schematic representation of the experimental setup detailing 660 nm illumination, input stimulation (1 Hz) in layer 4 barrel cortex, and recording of evoked fEPSPs in layer 3/4. Middle panel shows the induction of fEPSP potentiation by 660 nm light and in the presence of PKA inhibitor (KT5720), NMDA receptor blockers (5,7 DCK, MK801, and AP5). Right panel shows a summary of the effects on the fEPSP slope. (C) 660 nm light evoked fEPSP in cortical slices expressing DdPAC in WT, IP 3 R2 −/− and CalEX mice. Left panel shows a schematic representation of the astrocytic characteristics of the mouse lines used for MEA recording. Middle panel shows the induction of fEPSP slope potentiation by 660 nm light in the different mouse lines. Right panel shows a summary of the fEPSP slope potentiation effects. (D) 660 nm light evoked field potentials in cortical slices expressing DdPAC in WT in the presence or absence of 1 Hz input during MEA recordings. Middle panel shows the induction of fEPSP slope potentiation by light. Right panel shows a summary of the fEPSP slope potentiation effects. For A–D, Middle panels show plots of fEPSP slope at experimental time points where symbols are mean ± SEM for experiments in separate slices (n = 4–5), with symbols corresponding to indicated experimental conditions. Panels on the right show summary bar graphs of mean ± SEM (n = 4–5) of normalised fEPSP slope 60 min post stimulation, where each point represents data from a single slice. For comparison between LTP and baseline in MEA recordings, paired Student’s t test was used (∗∗ p < 0.01). Differences between the two independent groups were determined by the Mann-Whitney test (∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: PKA inhibitor KT5720 , Hello Bio: https://hellobio.com/kt-5720.html , HB0361.

Techniques: Expressing, Comparison, MANN-WHITNEY

Journal: Cells

Article Title: Inhibition of Vascular Smooth Muscle Cell Proliferation by ENPP1: The Role of CD73 and the Adenosine Signaling Axis.

doi: 10.3390/cells13131128

Figure Lengend Snippet: Figure 6. Effect of AMP on activation of Gs-coupled adenosine receptors and PKA. Synthetic VSMCs were pretreated for 18 h with either (a) dual A2aR/A2bR antagonist AB928 (0.3 µM) or (b) selective A2aR antagonist SCH-58261 (1 µM) and selective A2bR antagonist PSB-1115 (1 µM). Cells were washed and stimulated for 4 h with AMP in the presence or absence of inhibitors. Total cAMP was measured using HTRF assay. VSMCs were preincubated for 30 min in the basal media supplemented with 0.25% FBS and stimulated for 30 min with AMP in the absence (c) or presence (d) of PKA inhibitor KT5720. Phosphorylation of VASP at Ser157 was analyzed using HTRF assay. Values are presented as the mean ± SD, n = 4–6, ** p < 0.01, *** p < 0.001 (t-test).

Article Snippet: ATP, PKA inhibitor KT5720, A2aR antagonist SCH-58261, and A2bR antagonist PBS-1115 were from Tocris Biosciences (Minneapolis, MN, USA).

Techniques: Activation Assay, HTRF Assay, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2

doi: 10.1016/j.jbc.2023.104782

Figure Lengend Snippet: PIEZO2-dependent stretch-activated currents are not regulated by PKA. A , cartoon depicting the recording paradigm ( left ) and representative PIEZO2 stretch-activated current ( right ) evoked by increasing negative pressure pulses applied to N2a-P1KO cell patches in the absence ( black ) or the presence of the PKA inhibitor KT5720 ( gray ) and the PKA activator 8-Br-cAMP ( green ). See Fig. S2 for additional example traces. B , pressure–response curves ( left ) and scatter plot ( right ) of the maximal total charge transfer (area under the curve [AUC]) evoked by negative pressure of GFP empty vector or PIEZO2 from treated and untreated cells. Data are presented as the mean ± SD. Number of cells per group is indicated in the legend. Comparison with two-way ANOVA p = 0.7147. C , proportion of cells responding to pressure-induced membrane stretch amongst treated or untreated cells. The numbers of tested cells are indicated within the bars. Number of responders correspond to the ones indicated in the legend of ( B ). Fisher’s exact test, p = 0.0894 CTL versus KT, p = 0.7753 CTL versus 8Br, p = 0.1269 KT versus 8Br. D , example traces ( left ) of stretch-activated PIEZO2 currents from untreated ( black ), KT5720-treated ( gray ), and 8-Br-cAMP-treated ( green ) cells and shown with their corresponding current amplitude distribution histograms ( right ). The peak values of Gaussian fits ( purple ) representing the unitary current are indicated. E , linear regression fits of the I/V plots (unitary currents versus holding potential) of PIEZO2-treated and PIEZO2-untreated cells. Data are presented as the mean ± SD. Number of cells per group is indicated in the legend. F , unitary conductance of PIEZO2 in treated and untreated cells. Data are presented as the mean ± SEM with individual values. Number of cells per group is indicated above each bar. Comparison with Kruskal–Wallis p = 0.1341. 8-Br-cAMP, 8-bromo-cyclic-AMP.

Article Snippet: Cells are then used within 24 h to 48 h. To investigate PKA activity, N2a-P1KO cells were incubated the day before the experiments (patch clamp or imaging) with PKA inhibitor KT5720 (Sigma) and PKC inhibitor GF109203X (Sigma), both dissolved in dimethyl sulfoxide and used at a final concentration of 1 μM.

Techniques: Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2

doi: 10.1016/j.jbc.2023.104782

Figure Lengend Snippet: Mutation of predicted PKA sites from individual intracellular domain of PIEZO2 does not prevent PKA-dependent modulation. A , side view of the mouse PIEZO2 structure (Protein Data Bank ID: 6KG7 ) with the modeled intracellular disordered loops that contained the predicted high ( black spheres ) and low ( gray spheres ) scores. PKA phosphorylation sites colored in the indicated color. See also Table S1 . B , representative example traces from the different PIEZO2 mutants S387A/S412A ( top left ), the S1517A/S1633A ( top right ), the S1758A ( bottom left ), and the S2137A/S2318A/S2169A/S2190A ( bottom right ) in the presence of the PKA inhibitor KT5720 ( gray ) and the PKA activator 8-Br-cAMP ( green ). C – F , displacement–response curves of peak current amplitudes of PIEZO2 ( black ) and the different PIEZO2 PKA site mutants ( gray and green ) for cells treated with PKA inhibitor or activator. Data are presented as the mean ± SD. Number of cells per group is indicated in the legend. Comparison with Mann–Whitney test p < 0.05∗, p < 0.001∗∗, p < 0.0001∗∗∗, mutant + KT versus mutant + 8Br. G – I , comparison of the mean ± SD maximal current amplitude ( G ), mechanical activation thresholds ( H ), and inactivation time constants ( I ) of whole-cell currents from PIEZO2 mutants (same mutants as in B – F , labels only contain first mutation for better reading) treated with KT5720 ( gray ) or 8-Br-cAMP ( green ). Black circles represent individual data points. Number of cells per group are identical to those in C – F . Thresholds of the S387A/S412A mutant were compared with Mann–Whitney test p = 0.3550 and with Student's t test for all other mutants (∗ p = 0.0161, S1517A; ∗∗∗ p = 0.00017, S1758A; p = 0.0560, S2137A). 8-Br-cAMP, 8-bromo-cyclic-AMP.

Article Snippet: Cells are then used within 24 h to 48 h. To investigate PKA activity, N2a-P1KO cells were incubated the day before the experiments (patch clamp or imaging) with PKA inhibitor KT5720 (Sigma) and PKC inhibitor GF109203X (Sigma), both dissolved in dimethyl sulfoxide and used at a final concentration of 1 μM.

Techniques: Mutagenesis, MANN-WHITNEY, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2

doi: 10.1016/j.jbc.2023.104782

Figure Lengend Snippet: IDR5 del -mediated currents are not modulated by PKA. A , topological representation of PIEZO2 with its major domains, its intracellular intrinsically disordered regions (IDR1–7), and the location of the predicted PKA phosphorylation sites ( black and gray circles ). B , representative example traces of whole-cell current evoked by increasing mechanical indentation from PIEZO2 ( left ) and the previously characterized IDR5 del PIEZO2 mutant ( right ) in the presence of the PKA inhibitor KT5720 or the PKA activator 8-bromo-cAMP. C, displacement–response curves of peak current amplitudes of PIEZO2 and the indicated IDR del mutants after inhibition and activation of PKA. Data are presented as the mean ± SD. Number of cells per group is indicated in the legend. Comparison with Mann–Whitney test, p < 0.05∗, p < 0.001∗∗, p < 0.0001∗∗∗ 8-Br-cAMP versus KT. D and E , inactivation time constant (τ inact ) ( D ) and mechanical activation thresholds ( E ) of whole-cell current from PIEZO2 and IDR del mutants treated with KT5720 ( filled bars ) or 8-Br-cAMP ( dashed bars ). Data are presented as the mean ± SD with individual values. Number of cells per group are identical to those in ( C ). Inactivation time constants and activation thresholds of KT5720- and 8-Br-cAMP-treated cells were compared pairwise for each mutant using Mann–Whitney test. τ inact : PIEZO2, p = 0.000081; IDR1 del p = 0.0020. Activation threshold: PIEZO2, p = 0.004; IDR3 del p = 0.0018; IDR6 del p = 0.012. 8-Br-cAMP, 8-bromo-cyclic-AMP.

Article Snippet: Cells are then used within 24 h to 48 h. To investigate PKA activity, N2a-P1KO cells were incubated the day before the experiments (patch clamp or imaging) with PKA inhibitor KT5720 (Sigma) and PKC inhibitor GF109203X (Sigma), both dissolved in dimethyl sulfoxide and used at a final concentration of 1 μM.

Techniques: Mutagenesis, Inhibition, Activation Assay, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2

doi: 10.1016/j.jbc.2023.104782

Figure Lengend Snippet: PIEZO2 cluster size and density is not affected by PKA modulation. A , representative TIRF images of N2A-P1KO cells transfected with PIEZO2mScarlet and incubated without ( left , untreated) or with the PKA inhibitor KT5720 ( middle ) and the PKA activator 8-Br-cAMP ( right ). Scale bar is indicated in the images. B , average cluster densities of PIEZO2mScarlet in cells untreated (CTL, black or treated with KT5720 ( gray ) or 8-Br-cAMP ( green ). Data are presented as the mean ± SD with individual values. Number of cells is indicated in the graph. Comparison with one-way ANOVA, p = 0.5705. C , close-up view of the Gaussian fit of a PIEZO2mScarlet cluster ( left ) and average cluster size (in micrometer) per cell ( right ) and per treatment condition. Data are presented as the mean ± SD with individual values. Number of cells is indicated in the graph. Overall cluster number for CTL N = 3623, KT5720 N = 3792, and 8Br N = 5028. Scale bar is indicated in the image. Comparison with one-way ANOVA, p = 0.1066. See also Fig. S3 . D , close-up view and representative examples ( left ) of the four different trajectories observed for PIEZO2mScarlet clusters: confined ( cyan , top left ), subdiffusion ( orange , top right ), normal diffusion ( red , bottom left ), and directed ( green , bottom right ). Average proportion of the four defined PIEZO2 cluster trajectories per cell ( right ). Data are presented as the mean ± SD. Number of cells are identical to the ones in B and C . Overall track numbers are identical to those in Fig. S3 B . Scale bar represents 1 μm. Comparison with Kruskal–Wallis test, confined p = 0.4990, normal diffusion p = 0.1799, directed p = 0.5502, and subdiffusion p = 0.2792. E , representative fluorescent images ( inverted ) of N2A-P1KO cells untreated (control, left ) or treated with the PKA inhibitor KT5720 (KT, middle ) and the PKA activator 8-Br-cAMP (8Br, right ). Scale bar is indicated in the images. F , quantification of the N2A-P1KO cell area (in μm 2 ) incubated with the different PKA-modulated conditions. Data are presented as violin plot with the median value and the 25th and 75th quartile. Number of cells per group is CTL N = 1204, KT5720 N = 1637, and 8Br N = 939. Comparison with Kruskal–Wallis test p < 0.0001 and Dunn’s post-test, p = 0.00000065 CTL versus KT5720, p < 0.0001 KT versus 8Br, and p < 0.0001 CTL versus 8Br. 8-Br-cAMP, 8-bromo-cyclic-AMP; TIRF, total internal reflection microscopy.

Article Snippet: Cells are then used within 24 h to 48 h. To investigate PKA activity, N2a-P1KO cells were incubated the day before the experiments (patch clamp or imaging) with PKA inhibitor KT5720 (Sigma) and PKC inhibitor GF109203X (Sigma), both dissolved in dimethyl sulfoxide and used at a final concentration of 1 μM.

Techniques: Transfection, Incubation, Diffusion-based Assay, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2

doi: 10.1016/j.jbc.2023.104782

Figure Lengend Snippet: PIEZO1 mechanically activated currents evoked by membrane indentation are not affected by PKA modulation. A , side view of the full-length mouse PIEZO1 AlphaFold structure (E2JF22) with the intracellular disordered loops ( colored domains ) that contained the predicted high ( black sphere ) and low ( gray sphere ) score PKA phosphorylation sites. See also Table S2 . B , representative example traces from PIEZO1 untreated ( black , left ) or treated with KT5720 ( gray , middle ) and 8-Br-cAMP ( green , right ). C , displacement–response curves ( left ) and scatter plot of the maximal ( right ) peak current amplitudes of PIEZO1 untreated ( black ) and treated with PKA inhibitor KT5720 ( gray ) or activator 8-Br-cAMP ( green ). Data are presented as the mean ± SD. Number of cells per group is indicated in the legend. D , mechanical activation thresholds from PIEZO1-treated and -untreated cells. Data are presented as the mean ± SD with individual values. Number of cells are identical to ( B ). Comparison with one-way ANOVA, p = 0.3858. E , inactivation time constants (τ inact ) of PIEZO1-treated and -untreated cells. Data are presented as the mean ± SD with individual values. Number of cells are identical to ( B ). Comparison with Kruskal–Wallis test, p = 0.027 and Dunn’s post-test, p = 0.0266 CTL versus KT5720. 8-Br-cAMP, 8-bromo-cyclic-AMP.

Article Snippet: Cells are then used within 24 h to 48 h. To investigate PKA activity, N2a-P1KO cells were incubated the day before the experiments (patch clamp or imaging) with PKA inhibitor KT5720 (Sigma) and PKC inhibitor GF109203X (Sigma), both dissolved in dimethyl sulfoxide and used at a final concentration of 1 μM.

Techniques: Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2

doi: 10.1016/j.jbc.2023.104782

Figure Lengend Snippet: Simultaneous disruption of all the major predicted PKA sites of PIEZO2 disrupts PKA modulation of mechanically activated currents evoked by membrane indentation. A , side view of one protomer of the mouse PIEZO2 structure (Protein Data Bank ID: 6KG7 ) with the modeled intracellular disordered loops that contained the predicted high score PKA phosphorylation sites that are simultaneously mutated to alanine ( black spheres ). B , representative example traces from PIEZO2 ( left , black ) and the ninefold mutant (9MUT, right ) treated with PKA inhibitor KT5720 or with the PKA activator 8-Br-cAMP. C , displacement–response curves ( left ) and scatter plot of the maximal ( right ) peak current amplitudes of PIEZO2 ( black ) and PIEZO2-9MUT ( blue ), treated with PKA inhibitor KT5720 or activator 8-Br-cAMP. Data are presented as the mean ± SD. Number of cells per group is indicated in the legend. Comparison with Mann–Whitney test, p < 0.05∗, p < 0.001∗∗, p < 0.0001∗∗∗ PIEZO2 KT5720 versus 8Br. D , mechanical activation thresholds from PIEZO2 and PIEZO2-9MUT-treated cells with KT5720 ( filled bars ) and 8Br ( dashed bars ). Data are presented as the mean ± SD with individual values. Number of cells are identical to ( C ). Comparison with unpaired t test, p = 0.0166, PIEZO2 KT5720 versus 8Br, p = 0.9753 9MUT KT5720 versus 8Br. E , inactivation time constants (τ inact ) of PIEZO2- and PIEZO2-9MUT-treated cells with KT5720 ( filled bars ) and 8Br ( dashed bars ). Data are presented as the mean ± SD with individual values. Number of cells are identical to ( C ). Comparison with Mann–Whitney test, p = 0.3579, PIEZO2 KT5720 versus 8Br, p = 0.3562 9MUT KT5720 versus 8Br. 8-Br-cAMP, 8-bromo-cyclic-AMP.

Article Snippet: Cells are then used within 24 h to 48 h. To investigate PKA activity, N2a-P1KO cells were incubated the day before the experiments (patch clamp or imaging) with PKA inhibitor KT5720 (Sigma) and PKC inhibitor GF109203X (Sigma), both dissolved in dimethyl sulfoxide and used at a final concentration of 1 μM.

Techniques: Mutagenesis, MANN-WHITNEY, Activation Assay