Journal: Nature Communications

Article Title: Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

doi: 10.1038/s41467-025-67232-3

Figure Lengend Snippet: a Cryo-EM densities for Ca 2+ and NS309 bound to one CaM molecule in NS309_K Ca 2.2 are shown as blue and green mesh contoured at σ = 6, respectively. b Cryo-EM densities for Ca 2+ and NS309 bound to one CaM molecule in NS309_K Ca 3.1 are shown as magenta mesh contoured at σ = 6. c Intracellular view of NS309_K Ca 2.2 (K Ca 2.2: purple cartoon/CaM: light blue surface). The CaM N-lobes are positioned far apart, and the HC helices are not visible probably due to flexibility. d Intracellular view of NS309_K Ca 3.1 (K Ca 3.1: salmon cartoon/CaM: cyan surface). The CaM N-lobes are positioned close to each other, which stabilize the HC helices in the center.

Article Snippet: NS309 (6,7-dichloro-1 H -indole-2,3-dione 3-oxime) was purchased from Alomone labs. NS309 dilutions were prepared freshly in extracellular solution from 20 mM stock solutions in DMSO.

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

doi: 10.1038/s41467-025-67232-3

Figure Lengend Snippet: a Binding energy between NS309 and amino acid residues in CaM and the S 45 A helix of K Ca 2.2. b Binding energy between NS309 and amino acid residues in CaM and the S 45 A helix of K Ca 3.1. The binding energy between NS309 and the four subunits of the activator-bound structure include van der Waals forces (VDW, black) and electrostatic interactions (Electrostatic, red). Data are presented as mean ± SD (n = 4 channel subunits). c Responses of WT and mutant K Ca 2.2 channels to NS309 in whole-cell patch-clamp recordings in the presence of 0.25 μM Ca 2+ . d Responses of WT and mutant K Ca 3.1 channels to NS309 in whole-cell patch clamp recordings in the presence of 0.25 μM Ca 2+ . Data are presented as mean ± SD (n = 4 transfected cells). e Total binding energy of NS309 to binding pockets in NS309_K Ca 2.2 and NS309_K Ca 3.1, including van der Waals forces (VDW, black) and electrostatic interactions (Electrostatic, red). Data are presented as mean ± SD (n = 4 channel subunits). f Chemical structure of NS309.

Article Snippet: NS309 (6,7-dichloro-1 H -indole-2,3-dione 3-oxime) was purchased from Alomone labs. NS309 dilutions were prepared freshly in extracellular solution from 20 mM stock solutions in DMSO.

Techniques: Binding Assay, Mutagenesis, Patch Clamp, Transfection

Journal: Nature Communications

Article Title: Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

doi: 10.1038/s41467-025-67232-3

Figure Lengend Snippet: a Chemical structures of rimtuzalcap and CyPPA. b The binding pocket of rimtuzalcap in rimtuzalcap_K Ca 2.2_I (K Ca 2.2: green/CaM: yellow) superimposed onto the binding pocket of NS309 in NS309_K Ca 2.2 (K Ca 2.2: purple/CaM: light blue). Rimtuzalcap forms contacts with both the S 45 A and HA helices, while NS309 primarily interacts with the S 45 A helix. c Binding energy between rimtuzalcap and amino acid residues in CaM and the S 45 A helix of K Ca 2.2. The binding energy between rimtuzalcap and the four subunits of the activator-bound structure include van der Waals forces (VDW, black) and electrostatic interactions (Electrostatic, red). Data are presented as mean ± SD (n = 4 channel subunits).

Article Snippet: NS309 (6,7-dichloro-1 H -indole-2,3-dione 3-oxime) was purchased from Alomone labs. NS309 dilutions were prepared freshly in extracellular solution from 20 mM stock solutions in DMSO.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

doi: 10.1038/s41467-025-67232-3

Figure Lengend Snippet: In side views, dimensions of the inner gate are measured as distances between Val391 in the transmembrane S6 helices of opposite K Ca 2.2 subunits in a apo_K Ca 2.2, b NS309_K Ca 2.2, c rimtuzalcap_K Ca 2.2_I, and d AP14145_K Ca 2.2 structures. Two opposite channel subunits are shown for clarity.

Article Snippet: NS309 (6,7-dichloro-1 H -indole-2,3-dione 3-oxime) was purchased from Alomone labs. NS309 dilutions were prepared freshly in extracellular solution from 20 mM stock solutions in DMSO.

Techniques:

Journal: iScience

Article Title: Context-dependent NMDA receptor dysfunction predicts seizure treatment in mice with human GluN1 variant

doi: 10.1016/j.isci.2025.114301

Figure Lengend Snippet: Potentiating SK channels restores normal NMDAR integration in Grin1 Y647S +/− mice (A) Schematic of potential mechanism showing impaired negative feedback in Y647S +/− neurons due to insufficient Ca 2+ influx compared to WT and the effect of NS309 in boosting SK channel Ca 2+ sensitivity, thereby restoring negative feedback in Y647S +/− neurons. (B) Average NMDAR plateau potential in Y647S +/− neurons at 70 μA with extended tail indicated by red arrow. Application of 10 μM NS309 to the slice restores normal duration and terminates the NMDAR plateau potential in Y647S +/− (Y647S +/− + NS309). (Inset) Restoration of plateau potential duration by NS309 at increasing stimulus intensities in a Y647S +/− neuron. (C) Total width of the NMDAR plateau potential in WT, Y647S +/− , and Y647S +/− + NS309 neurons (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 10 −4 , Tukey’s post hoc. Black stars: WT vs. Y647S +/− , purple stars: Y647S +/− vs. Y647S +/− + NS309). (D) SK2 channel blocker (Leidab7, 100 nM) prevents NS309 from reducing plateau potential duration in Y647S +/− neurons. (E) Normalized NMDAR plateau width in Y647S +/− neurons with the addition of NS309 and NS309+SK2 blockers (∗ p < 0.05, Sidak’s post hoc).

Article Snippet: NS309 , Tocris , 3895.

Techniques:

Journal: iScience

Article Title: Context-dependent NMDA receptor dysfunction predicts seizure treatment in mice with human GluN1 variant

doi: 10.1016/j.isci.2025.114301

Figure Lengend Snippet: Potentiating SK channels restores normal NMDAR integration in Grin1 Y647S +/− mice (A) Schematic of potential mechanism showing impaired negative feedback in Y647S +/− neurons due to insufficient Ca 2+ influx compared to WT and the effect of NS309 in boosting SK channel Ca 2+ sensitivity, thereby restoring negative feedback in Y647S +/− neurons. (B) Average NMDAR plateau potential in Y647S +/− neurons at 70 μA with extended tail indicated by red arrow. Application of 10 μM NS309 to the slice restores normal duration and terminates the NMDAR plateau potential in Y647S +/− (Y647S +/− + NS309). (Inset) Restoration of plateau potential duration by NS309 at increasing stimulus intensities in a Y647S +/− neuron. (C) Total width of the NMDAR plateau potential in WT, Y647S +/− , and Y647S +/− + NS309 neurons (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 10 −4 , Tukey’s post hoc. Black stars: WT vs. Y647S +/− , purple stars: Y647S +/− vs. Y647S +/− + NS309). (D) SK2 channel blocker (Leidab7, 100 nM) prevents NS309 from reducing plateau potential duration in Y647S +/− neurons. (E) Normalized NMDAR plateau width in Y647S +/− neurons with the addition of NS309 and NS309+SK2 blockers (∗ p < 0.05, Sidak’s post hoc).

Article Snippet: SK channel positive allosteric modulator NS309 (10 μM, Tocris ) was included in a subset of experiments to restore appropriate duration of dendritic integration in Y647S +/− mice.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Activation of Small Conductance Ca 2+ -Activated K + Channels Suppresses Electrical and Calcium Alternans in Atrial Myocytes

doi: 10.3390/ijms26083597

Figure Lengend Snippet: Effect of SK channel inhibition and activation on atrial APD. ( A ): ( a ) Overlay of APs recorded from the same atrial myocyte in control and after application of SK channel blocker apamin (100 nM) at 1 Hz stimulation. ( b ) Mean ± SD and individual APD 70 and APD 90 values in control (Ctrl) and in apamin (n/N = 15/10; paired t -test). ( B ): ( a ) Overlay of APs recorded from the same cell in control and after application of SK channel activator NS309 (2 µM). ( b ) Mean ± SD and individual APD 70 and APD 90 measurements in control and after application of NS309 (n/N = 6/2; paired t -test).

Article Snippet: 10 mM NS309 (Cayman Chemical, Ann Harbor, MI, USA), 5 mM UCL1684 (Tocris/Bio-Techne, Minneapolis, MN, USA), and 5 mM HMR1556 (MilliporeSigma, St. Louis, MO, USA) stock solutions were prepared in DMSO.

Techniques: Inhibition, Activation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Activation of Small Conductance Ca 2+ -Activated K + Channels Suppresses Electrical and Calcium Alternans in Atrial Myocytes

doi: 10.3390/ijms26083597

Figure Lengend Snippet: Effect of SK channel activation on CaT alternans. ( A ): CaTs recorded from the same atrial myocyte in control, in the presence of SK channel activator NS309 (2 µM) and after washout of NS309 (stimulated at 1.6 Hz). CaT alternans were induced by increasing field stimulation frequency. ( B ): Mean ± SD and individual cell CaT AR values in control during NS309 exposure (n/N = 17/6) and after washout in a subset of cells (n/N = 9/3). Statistical analysis was performed using Tukey’s mixed-effects groups comparison test.

Article Snippet: 10 mM NS309 (Cayman Chemical, Ann Harbor, MI, USA), 5 mM UCL1684 (Tocris/Bio-Techne, Minneapolis, MN, USA), and 5 mM HMR1556 (MilliporeSigma, St. Louis, MO, USA) stock solutions were prepared in DMSO.

Techniques: Activation Assay, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Activation of Small Conductance Ca 2+ -Activated K + Channels Suppresses Electrical and Calcium Alternans in Atrial Myocytes

doi: 10.3390/ijms26083597

Figure Lengend Snippet: Activation of SK channels abolishes APD alternans. ( A ): APs recorded from the same current-clamped atrial myocyte stimulated at 2 Hz in control and in the presence of SK channel activator NS309 (2 µM). ( B ): Mean ± SD and individual APD 70 and APD 90 ratios of pairs of alternating APs in control and in the presence of NS309 (n/N = 6/4). A dashed line marks a ratio of 1, indicating the absence of alternans. Statistical analysis was performed by paired t -test.

Article Snippet: 10 mM NS309 (Cayman Chemical, Ann Harbor, MI, USA), 5 mM UCL1684 (Tocris/Bio-Techne, Minneapolis, MN, USA), and 5 mM HMR1556 (MilliporeSigma, St. Louis, MO, USA) stock solutions were prepared in DMSO.

Techniques: Activation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Activation of Small Conductance Ca 2+ -Activated K + Channels Suppresses Electrical and Calcium Alternans in Atrial Myocytes

doi: 10.3390/ijms26083597

Figure Lengend Snippet: The inhibition of SK channels abolishes NS309’s effects on APD and CaT alternans. ( A ): ( a ) Overlay of APs recorded at 1 Hz from the same atrial myocyte in control, in apamin (100 nM), and in apamin together with NS309 (2 µM). ( b ) Changes in APD 70 and APD 90 compared to control (dashed lines) in the presence of NS309 alone (n/N = 15/10) and in the presence of NS309 + apamin (n/N = 9/5). Mean ± SD and individual cell measurements are shown. Cells were pretreated with apamin for 3 min before measurements. Statistical analysis was performed using the Welsh t -test. ( B ): ( a ) CaTs recorded from the same field stimulated (2.8 Hz) atrial myocytes in control, in the presence of apamin, during simultaneous application of NS309 and apamin and in NS309 alone. ( b ) Mean ± SD and individual CaT ARs recorded using the experimental protocol from panel Ba (n/N = 9/5); Tukey’s multiple group comparison test.

Article Snippet: 10 mM NS309 (Cayman Chemical, Ann Harbor, MI, USA), 5 mM UCL1684 (Tocris/Bio-Techne, Minneapolis, MN, USA), and 5 mM HMR1556 (MilliporeSigma, St. Louis, MO, USA) stock solutions were prepared in DMSO.

Techniques: Inhibition, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Activation of Small Conductance Ca 2+ -Activated K + Channels Suppresses Electrical and Calcium Alternans in Atrial Myocytes

doi: 10.3390/ijms26083597

Figure Lengend Snippet: Effect of SK channel activation on CaT amplitude. ( A ): Overlay of CaTs recorded from the same field stimulated (0.5 Hz; no alternans) atrial myocyte in control (black), in NS309 (red), and after washout of NS309 (grey). ( B ): Mean ± SD and individual values of CaT amplitudes (ΔF/F 0 ) recorded in control, in the presence of NS309 and during subsequent application of NS309 together with two different SK channel blockers: ( a ) apamin (n/N = 11/3) and ( b ) UCL1684 (1 µM; n/N = 9/3). Statistical analysis was performed using Tukey’s multiple-group comparison test. ( C ): Mean ± SD and individual values of CaT amplitudes observed in control and in the presence of 100 nM of apamin (n/N = 6/2), demonstrating that blocking SK channels had no effect on CaT amplitude. ( D ): ( a ) Caffeine (10 mM) induced CaTs recorded in the same atrial cell (stimulated at 0.5 Hz) before and after NS309 application, shown at different time scales. The top trace shows the decrease in CaT amplitude over time in the presence of NS309. ( b ) Mean ± SD and individual values of caffeine-induced CaT amplitudes (ΔF/F 0 ) recorded in control and in the presence of NS309 (n/N = 8/3; paired t -test).

Article Snippet: 10 mM NS309 (Cayman Chemical, Ann Harbor, MI, USA), 5 mM UCL1684 (Tocris/Bio-Techne, Minneapolis, MN, USA), and 5 mM HMR1556 (MilliporeSigma, St. Louis, MO, USA) stock solutions were prepared in DMSO.

Techniques: Activation Assay, Control, Comparison, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Activation of Small Conductance Ca 2+ -Activated K + Channels Suppresses Electrical and Calcium Alternans in Atrial Myocytes

doi: 10.3390/ijms26083597

Figure Lengend Snippet: Activation of SK channels reduces the risk for CaT alternans in atrial myocytes with simulated LQT syndrome type-1. ( A ): ( a ) Overlay of APs recorded at 1 Hz from the same atrial myocyte in control and after application of Kv7.1 potassium channel blocker HMR1556 (1 µM) simulating LQT syndrome type-1 conditions. ( b ) Mean ± SD and individual APD 70 and APD 90 values recorded in control and in the presence of HMR1556 (n/N = 8/5; paired t -test). ( B ): ( a ) CaTs recorded from the same field stimulated (1.8 Hz) atrial myocyte in control, in HMR1556, and during simultaneous application of HMR1556 and NS309. ( b ) Mean ± SD and individual CaT AR values recorded in control, in the presence of HMR1556 (n/N = 14/8), followed by simultaneous application of HMR1556 and NS309 (n/N = 13/7). Statistical analysis with Tukey’s mixed-effects group comparison test.

Article Snippet: 10 mM NS309 (Cayman Chemical, Ann Harbor, MI, USA), 5 mM UCL1684 (Tocris/Bio-Techne, Minneapolis, MN, USA), and 5 mM HMR1556 (MilliporeSigma, St. Louis, MO, USA) stock solutions were prepared in DMSO.

Techniques: Activation Assay, Control, Comparison