Lipid interactions with the R/K-rich region, movement of the CTD, and open state stability. (A) Interactions between negatively charged PIP₂ and positively charged R/K residues are preserved during channel activation and pore opening (Sim. 3); PIP₂ atoms within 4 Å of an R/K residue were defined as interacting. (B) Per-subunit (i.e., A–D) distribution of POPS:P distances to K62:Cζ/R65:Nζ (IH), R78:Nζ/R80:Nζ (TM1), and R219:Nζ/K220:Cζ (CTD). (C) Snapshot of one TM subunit (gray) and part of the CTD of an adjacent subunit (green) highlighting PIP₂ and POPS-interacting (R/K) residues. SF-bound ions are shown as colored spheres. (D) CTD-SF center of mass separation during pore opening in simulations of the activation gate G178R mutant (Sim. 3) and upon back mutation of this mutant to the WT (Sim. 4). Green dotted lines are reference distances from the apo (PDB ID 3JYC) and PIP₂-bound (PDB ID 3SPI) crystal structures (Tao et al., 2009; Hansen et al., 2011). Inset: Two crystal structures and a simulation snapshot of the open state obtained in Sim. 4; dotted red line indicates the CTD movement toward the TM domains (all structures were TM-aligned). (E) Stability of the open pore conformation (Sim. 4) assessed by measuring the C_α–C_α diagonal distance at activation gate residues I177 and M181 (top), the pore cavity hydration, and the CTD-SF center of mass separation (bottom).