Structural mechanisms for inhibition and activation of human small-conductance Ca2+-activated potassium channel SK2

Fig. 1. Cryo-EM structure of human SK2 in complex with CaM. a Cryo-EM density map of the human SK2-CaM complex viewed from the membrane plane (left), extracellular side (middle), and cytosol (right). Four promoters of SK2 are shown in pink, orange, purple, and cyan, and CaM in yellow. b Cartoon representation of SK2-CaM complex. The same colour scheme from panel a is applied. Calcium ions and lipids are depicted in green spheres and gray sticks, respectively. c The binding of CaM to one protomer of SK2. CaM interacting with the S4–S5 linker and the HA helix is colored in gray and yellow, respectively. d Superposition of SK2 (pink) and SK4 (gray, PDB ID: 6cnn). Detailed interactions between S3 and S4 helices are shown on the right. A cholesterol-like density in SK2 is shown in green meshes. DDM in SK4 is depicted in wheat sticks.

Fig. 2. Structural basis for SK2 blockade by apamin and UCL1684. a Sequence of apamin and the chemical structure of UCL1684. Arg13 and Arg14 of apamin are shown as red. b Concentration-dependent inhibition of SK2 by apamin. Left, representative current traces of SK2-expressing HEK293 cells. Data are normalized and presented as mean values ± SEM (n = 8 cells). c Cryo-EM density map of SK2-apamin. SK2 in gray, apamin in red. d Apamin binds to the outer vestibule of SK2. Residues involved in apamin binding are shown as sticks. Apamin in red, SK2 in green, purple, orange, and pink, respectively. K+ ions are depicted in blue spheres. e Interactions between apamin and the S3–S4 loop. Black dashed lines indicate polar interactions. f UCL1684 binds in the outer vestibule of SK2. UCL1684 is shown as yellow sticks. g Inhibition of SK2 variants by apamin and UCL1684. Data are shown as mean ± SEM (n = 6 cells) of normalized currents recorded from HEK293 cells expressing A242F, F243A, S244F, Y245A, H336N, and N367H in response to 300 nM apamin or 1 μM UCL1684. h Concentration-dependent inhibition curves of SK2 variants by apamin. Data were normalized and presented as mean values ± SEM (WT, n = 8;…

Fig. 3. Structural basis for SK2 potentiation by CAD-1883. a Chemical structure of CAD-1883. b Concentration-dependent activation curve of SK2 by CAD-1883. Representative whole-cell current traces of SK2 enhanced by CAD-1883. Voltage ramp, -100 mV to 100 mV (Left). Data are presented as mean values ± SEM (n = 8 cells). Curve was fitted using nonlinear regression. c Cryo-EM density map of CAD-1883 in SK2-CAD-1883 complex. Density maps for CAD-1883 and interacting residues are shown in purple and gray meshes, respectively. d Detailed interactions between CAD-1883 and SK2-CaM complex. CAD-1883 in cyan sticks, residues involved in CAD-1883 binding are shown in sticks. e–h CAD-1883 stimulated current traces of WT SK2 (e), L291W (f), L291F (g), and LN291-292AA (h). The free Ca2+ concentration in the internal solution is 300 nM. CAD-1883 is applied to the external solution at 100 μM. i Pore radius analysis of SK2-apamin and SK2-CAD-1883 by HOLE. Two opposing subunits are shown as cartoons; pores of SK2-apamin and SK2-CAD-1883 are shown as pink and yellow, respectively. j Pore radius profiles of SK2-apamin and SK2-CAD-1883 along the pore axis. Selectivity filter (SF) and activation gate (AG)…

Fig. 4. Structural basis for SK2 inhibition by AP30663. a Chemical structure of AP30663. b Concentration-dependent inhibition curve of SK2 by AP30663. Left, representative whole-cell current traces of SK2 inhibited by AP30663. Voltage ramp, −100 mV to 100 mV (Left). Data are presented as mean values ± SEM (n = 8 cells). Curve was fitted using nonlinear regression. c Cryo-EM structure of SK2-AP30663. SK2-AP30663 in pink sticks. d Cryo-EM density map of AP30663 in the central cavity of SK2-AP30663 (left). As a comparison, no such density is observed in SK2-apamin. Density maps for AP30663 and SK2 are shown in pink and gray, respectively. K+ ions are depicted as blue spheres. e Interactions between AP30663 and SK2 complex. Residues are indicated with black lines. f Inhibitory effects of 30 μM AP30663 on SK2 variants. Data are normalized and presented as mean values ± SEM (WT, n = 6; A383F, n = 5; A383L, n = 6; T386L, n = 7; T386F, n = 5 cells). g Representative whole-cell current trace of mutation A383L inhibited by AP30663, elicited by voltage ramps (−100 mV to +100 mV). h Concentration-dependent inhibition curve of SK2-A383L by AP30663. Data are presented as mean values ± SEM (n = 7…

Fig. 5. Proposed model of SK2 activation and modulation. a When intracellular Ca2+ is at low levels, the SK2 channel assumes a closed resting state. The C-lobe of CaM binds to the HA helix of SK2, while the N-lobe is dynamic. b After intracellular Ca2+ increased to sub-micromolar levels, Ca2+ ions bind to CaM N-lobe and induce conformational changes that facilitate the engagement of CaM N-lobe and the S45A helix, resulting in pore opening. c Multiple modulation sites in the SK2-CaM complex. Site 1 in the outer vestibule for apamin and UCL1684; Site 2 in the central cavity for AP30663 analogs; Site 3 in the CaM N-lobe and SK2 S45A helix interface for activators such as CAD-1883 and NS-309.