The structures of the undocked INX-6 hemichannels (WT in detergent, WT in a nanodisc, and INX-6ΔN in a nanodisc) were embedded with VMD 1.9.3 (57) in solvated lipid bilayers comprising 447 to 490 POPC molecules, ~100 mM KCl, and 72,000 to 76,000 water molecules. The spatial arrangement of the proteins in the lipid bilayer was determined by referring to the data of the docked INX-6 gap junction channel (PDB ID: 5H1R) in the OPM (orientation of protein in membrane) database. In the WT-in-detergent model, lipid molecules were removed from the pore and water molecules were placed there instead. In the WT-in-nanodisc and INX-6ΔN–in–nanodisc models, lipid molecules were retained in the pores. For the WT-in-detergent model, the system was first energy-minimized and then equilibrated at 300 K and 1 × 105 bar in three consecutive runs of 500-ps MD simulations. In the first run, only non-protein atoms were allowed to move. In the second and third runs, position restraints with a force constant of 1 kcal mol−1 Å−2 were imposed on all the protein atoms and on only the Cα atoms, respectively. Last, a 100-ns MD simulation was performed without restraints. For the WT-in-nanodisc model, only the atoms of the fatty acid parts of the lipid were allowed to move in the first equilibration run. In the second run, the position restraints were imposed on all the protein atoms and the phosphorus atoms of the lipids in the pore. In the third run, they were imposed on only the Cα atoms. After the equilibration MD runs, a space was formed in the lipid bilayer in the pore because the lipid molecules moved to form closer interactions with the protein or with other lipid molecules. Lipid molecules were inserted into the space from a preequilibrated lipid bilayer model, and water molecules that overlapped with the inserted lipid molecules were removed. The equilibration-insertion process was repeated three times. The system was equilibrated again, and a 100-ns unrestrained MD simulation was performed. For the INX-6ΔN–in–nanodisc model, the equilibration-insertion process was repeated four times. The system was equilibrated again, and an MD simulation was performed without restraints. We found, however, that a space was formed in the lipid bilayer in the pore in 20 ns. Therefore, we inserted lipid molecules into the space and equilibrated the system. Then, a 100-ns unrestrained MD simulation was performed.

The CHARMM36m force-field parameters (58) were used for the protein, lipid, and ions. The TIP3P model (59) was used for water. The temperature was controlled with the Langevin dynamics method (60, 61). The pressure was regulated with the Langevin piston method (62). Bond lengths involving hydrogen atoms were constrained using the SHAKE algorithm (63, 64) to allow the use of a large time step (2 fs). Electrostatic interactions were calculated with the particle mesh Ewald method (65, 66). All MD simulations were performed with NAMD 2.12 (67), with coordinates recorded every 10 ps.

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