PIP2 activation of the cardiac IKs potassium channel

Fig. 1. Conformations of KCNQ1 in various functional states. a Cryo-EM density map of KCNQ1-E160R/R231E IC state in the presence of PIP2. KCNQ1 and CaM are colored in blue and orange, respectively. The head and tail groups of PIP2 are colored red and black, respectively, to show its location. b Comparison of voltage sensor domains between KCNQ1-RC state (PDB: 8SIN), KCNQ1-I state (PDB: 8SIM), KCNQ1-AO state (PDB: 9VEO), and KCNQ1-E160R/R231E intermediate state in this study. Only the helical regions of S2-S4 and S4S5L are shown for clarity. The C-alpha atoms of the positive charged (or polar) resides on S4 are shown in yellow spheres with stick side chains, and the gating charge transfer center residues F167 (green), E170, and D202 are shown as sticks. Mutated residues are labeled with red color. c The ion conductance path and core radius for KCNQ1 bent conformation (PDB: 6UZZ), KCNQ1 with PIP2 IC bent conformation, and KCNQ1-KCNE1 with PIP2 AO straight conformation (PDB: 9VEI), respectively. Figures are plotted using the HOLE program. d Representative current traces of one interaction pairs between KCNQ1-S2S3L and CaM: KCNQ1-Y184W and CaM-D96R, displayed without (upper) and with (lower)…

Fig. 2. C-PIP2 binding is essential for channel activation. a C-PIP2 binding site in the KCNQ1-KCNE1-PIP2 complex (PDB: 9VEI). KCNQ1, KCNE1 and PIP2 are colored in blue, magenta and black, respectively. C-PIP2 is shown with ball-and-stick model. Orange color represents the Phosphorus (P) atoms, black represents Carbon (C) atoms, and red represents Oxygen (O) atoms. Residues including interactions with C-PIP2 are displayed. Key interactions are labeled with dashed lines. b Representative current recordings of KCNQ1-WT (left) and mutant KCNQ1-R259Q (middle) channels, displayed without (upper) and with (lower) KCNE1 co-expression. Right: G–V relationship comparison with WT channels. c Current amplitude comparison (+40 mV without KCNE1, +80 mV with KCNE1) of mutagenesis screens of all labeled C-PIP2 binding sites in (a). The black bars show without KCNE1, and the red bars show with KCNE1. d V1/2 of mutagenesis screens (black for without KCNE1, red for with KCNE1) of all labeled C-PIP2 binding sites in (a). e Structure depiction of the straight conformation of one monomer of tetrameric KCNQ1-KCNE1-CaM complex (PDB: 9VEI) in the presence of C-PIP2. KCNQ1, KCNE1, PIP2, and CaM are colored in…

Fig. 3. V-PIP2 binding is essential for channel activation to AO but not to IO. a V-PIP2 binding site in the KCNQ1-KCNE1-PIP2 complex (PDB: 9VEI). KCNQ1, KCNE1, and V-PIP2 are colored in blue, magenta, and black, respectively. Orange color represents the Phosphorus (P) atoms, black represents Carbon (C) atoms, and red represents Oxygen (O) atoms. Residues interaction with V-PIP2 are displayed. Key interactions are labeled with dashed lines. b Representative current recordings of KCNQ1-WT (left) and mutant KCNQ1-Y184W (middle) channels, displayed without (upper, black) and with (lower, red) KCNE1 co-expression. Right: G-V relationship comparison with WT channels. c Current amplitude comparison of mutagenesis screens (+40 mV without KCNE1, +80 mV with KCNE1) of all labeled V-PIP2 binding sites on (a). The black bars show without KCNE1, and the red bars show with KCNE1. d V1/2 of mutagenesis screens (black bars: without KCNE1, red bars: with KCNE1) of all labeled V-PIP2 binding sites in (a). e Representative current recordings of KCNQ1-WT + KCNE3-WT (black), mutant KCNQ1-Y184W (red) + KCNE3-WT and KCNQ1-K196Q (green) + KCNE3-WT channels. f The current amplitudes (at +40 mV) for the…

Fig. 4. V-PIP2 modulates IO to AO transition. a V-PIP2 binding pocket with key residues displayed for KCNQ1-E1R/R2E bent conformation (left, this study) and KCNQ1-KCNE1 straight conformation (right, PDB: 9VEI), respectively. KCNQ1, CaM, KCNE1, and V-PIP2 are colored in blue, orange, magenta, and black, respectively. Orange color represents the Phosphorus (P) atoms, black represents Carbon (C) atoms, and red represents Oxygen (O) atoms. Residues including interactions with V-PIP2 are displayed. Key interactions are labeled with dashed lines. b In silico screening of CA1 to the straight conformation of KCNQ1-KCNE1 (PDB: 9VEI) and the bent conformation of KCNQ1 (PDB: 6UZZ). KCNQ1, KCNE1, and CA1 are colored in tan, sky blue and red, respectively. The CA1 binding pocket is represented in orange. c CA1 effects on IKs channels. Current traces were recorded before (black) and after (red) adding 10 μM CA1. Average G-V relationship (lower) of IKs channels before (black) and after (red) adding 10 μM CA1. The EC50 of CA1 is 7.22 ± 0.57 μM (Fig. S13b). d Current amplitude comparative effects of CA1 on KCNQ1-WT (yellow), KCNQ1-WT + KCNE3-WT (green), IKs, and putative binding residues (rose pink) from…

Fig. 5. A model depicting the activation of KCNQ1 by VSD, C-PIP2, V-PIP2, and CaM. In this model, KCNQ1-RC, IC, and IO states adopt a bent conformation, while the AC and AO states adopt a straight conformation. V-PIP2 associates with the channel during activation of the VSD to the I state, including IC and IO, through a voltage-dependent mechanism that triggers the IO-AO transition. CaM and V-PIP2 compete for binding to the S2S3L; when CaM binds, the channel adopts a bent conformation, whereas binding of V-PIP2 results in a straight conformation. C-PIP2 interacts with the channel in both the I and A states, facilitating the coupling of VSD and pore during the IC-IO and AC-AO transitions.