Structural basis for the subtype-selectivity of KCa2.2 channel activators

Figures

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  Fig. 1. Different conformations of CaM in apo_KCa2.2 and apo_KCa3.1_II. a Side view of apo_KCa2.2 (KCa2.2: blue/CaM: gray, PDB 8V2G) shows channel-CaM interactions. The cytoplasmic HC helices are invisible. b Side view of apo_KCa3.1_II (KCa3.1: yellow/CaM: pink, PDB 6CNO) shows channel-CaM interactions. The C-terminal HC helices are visible. c Intracellular view of apo_KCa2.2 (KCa2.2: blue cartoon/CaM: gray surface). The CaM N-lobes are positioned far apart, and the HC helices are not visible probably due to flexibility. d Intracellular view of apo_KCa3.1_II (KCa3.1: yellow cartoon/CaM: pink surface). The CaM N-lobes are positioned close to each other, which stabilize the HC helices in the center.

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  Fig. 2. Conformation of CaM in activator-bound NS309_KCa2.2 and NS309_KCa3.1 structures. a Cryo-EM densities for Ca2+ and NS309 bound to one CaM molecule in NS309_KCa2.2 are shown as blue and green mesh contoured at σ = 6, respectively. b Cryo-EM densities for Ca2+ and NS309 bound to one CaM molecule in NS309_KCa3.1 are shown as magenta mesh contoured at σ = 6. c Intracellular view of NS309_KCa2.2 (KCa2.2: 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_KCa3.1 (KCa3.1: salmon cartoon/CaM: cyan surface). The CaM N-lobes are positioned close to each other, which stabilize the HC helices in the center.

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  Fig. 3. Calculated binding energy between NS309 and the binding pockets in NS309_KCa2.2 and NS309_KCa3.1. a Binding energy between NS309 and amino acid residues in CaM and the S45A helix of KCa2.2. b Binding energy between NS309 and amino acid residues in CaM and the S45A helix of KCa3.1. 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 KCa2.2 channels to NS309 in whole-cell patch-clamp recordings in the presence of 0.25 μM Ca2+. d Responses of WT and mutant KCa3.1 channels to NS309 in whole-cell patch clamp recordings in the presence of 0.25 μM Ca2+. Data are presented as mean ± SD (n = 4 transfected cells). e Total binding energy of NS309 to binding pockets in NS309_KCa2.2 and NS309_KCa3.1, 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.

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  Fig. 4. Conformation of CaM in the activator-bound rimtuzalcap_KCa2.2_I structure. a Cryo-EM densities for Ca2+ and rimtuzalcap bound to one CaM molecule are shown as blue and magenta mesh, respectively. The densities are contoured at σ = 6. b One Ca2+-bound CaM molecule of rimtuzalcap_KCa2.2_I (CaM: yellow/Ca2+: orange) superimposed on a Ca2+-bound CaM molecule of apo_KCa2.2 (CaM: gray/Ca2+: black) with the hydrophobic surfaces at the N- and C-lobes facing away. c One Ca2+-bound CaM molecule of rimtuzalcap_KCa2.2_I superimposed on a Ca2+-bound CaM molecule of apo_KCa2.2 with the hydrophobic surfaces at the N- and C-lobes facing the viewer. d Intracellular view of rimtuzalcap_KCa2.2_I (KCa2.2: green cartoon/CaM: yellow surface). Rimtuzalcap shortens the distances between the CaM N-lobes, which stabilize and make the HC helices visible.

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  Fig. 5. Calculated binding energy between rimtuzalcap and the binding pocket in Rimtuzalcap_KCa2.2_I. a Chemical structures of rimtuzalcap and CyPPA. b The binding pocket of rimtuzalcap in rimtuzalcap_KCa2.2_I (KCa2.2: green/CaM: yellow) superimposed onto the binding pocket of NS309 in NS309_KCa2.2 (KCa2.2: purple/CaM: light blue). Rimtuzalcap forms contacts with both the S45A and HA helices, while NS309 primarily interacts with the S45A helix. c Binding energy between rimtuzalcap and amino acid residues in CaM and the S45A helix of KCa2.2. 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).

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  Fig. 6. The sensitivity of KCa2.2 and KCa3.1 channels to rimtuzalcap. a Representative current traces of concentration-dependent activation of the KCa2.2_WT channel by rimtuzalcap in inside-out patch clamp recordings in the presence of 0.15 μM Ca2+. b Limited responses to rimtuzalcap of the KCa3.1_WT channel in inside-out patch clamp recordings in the presence of 0.15 μM Ca2+. c Representative current traces of concentration-dependent activation of the KCa3.1_R355K mutant channel by rimtuzalcap in inside-out patch clamp recordings in the presence of 0.15 μM Ca2+. d Responses of KCa3.1_R355K channels versus KCa2.2_WT channels to rimtuzalcap. Rimtuzalcap activates KCa3.1_R355K (EC50: 76.8 ± 21.3 μM, n = 8 transfected cells) with ~15-fold lower potency than KCa2.2_WT (EC50: 5.1 ± 1.0 μM, n = 5 transfected cells, P < 0.0001, unpaired two-tailed Student’s t-test). The responses were normalized by the maximal currents induced by 10 μM Ca2+. Data are presented as mean ± SD. e In rimtuzalcap_KCa3.1_R355K_I, cryo-EM densities for Ca2+ and rimtuzalcap bound to one CaM molecule are shown as magenta and blue mesh, respectively. The densities are contoured at σ = 6. f Binding energy between…

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  Fig. 7. Inner gate of KCa2.2 channels regulated by pharmacological agents. In side views, dimensions of the inner gate are measured as distances between Val391 in the transmembrane S6 helices of opposite KCa2.2 subunits in a apo_KCa2.2, b NS309_KCa2.2, c rimtuzalcap_KCa2.2_I, and d AP14145_KCa2.2 structures. Two opposite channel subunits are shown for clarity.