Overall structures of BK and Kv channels. a) The overall structure of BK channels in the Ca2+-free state (PDB: 6v3g) with key domains and regions labelled. Each monomer is shown in the same color. b) Top view of the TMD of BK channels, showing the non-domain- swapped VSD/PGD configuration. c) Top view of the TMD of Kv 1.2 channel (PDB: 3lut), where the VSDs and PGDs are domain-swapped. Note the much tighter packing of S4 from VSD and S5/S6 from PGD in BK channels.

Voltage activation of Core-MT BK channels.

a-d) z-displacement of key side chain charged groups from initial positions, z-displacement of centers-of-mass of VSD helices from initial positions, backbone RMSD of the pore-lining S6 (F307-L325) to the open state, and tilt angles of all TM helices during a 10-μs simulation under 750 mV (sim2b in Table S1). The locations of charged groups were taken as those of guanidinium CZ atoms (for Arg) and sidechain carboxyl carbons (for Asp/Glu). Each data point in a-d represents the average of four subunits for a given snapshot (thin grey lines), and the colored thick lines plot the running average. Only residues 313-324 of S6 were include in tilt angle calculation, and the values in the open and closed Cryo-EM structures are marked using purple dashed lines for reference in panel d. e) Superimposition of the initial (0 μs) and final (10 μs) structures of the pore (residues F307-L325; red cartoon), in comparison to the open Cryo-EM structure (cyan cartoon). The view shown is from the bottom (cytosolic side). f) Average pore profiles calculated from the first and last 0.1 μs of sim2b, with error bars showing standard error. The pore profile derived from PDB 5tj6 (open state) is shown as a reference.

Pore Rehydration and Ion Conductance.

a) The number of water molecules inside the pore as a function of time during simulation (sim 2b), with the upper panel showing recorded ion permeation events. Inserts show snapshots of the pore region at three representative timepoints. b) Snapshots illustrating key steps of K+ ions passing through the filter. Potassium ions inside or near the filter are colored according to their identities. The water molecule bridging two ions inside the filter is also shown as van der Waals spheres.

VSD gating charge and voltage-sensing movements.

a) Average voltage-induced movements of key charges along the membrane normal (z-axis) with respect to the initial resting state structure, derived from last 500 ns of 750 mV simulation sim 2b. Error bars show the standard deviations. b) Conformations of key charged residues in the resting (silver) and activated (orange) states of BK VSD. The resting and activated states are represented using the snapshots at 0 and 10 μs of sim2b, respectively. c) Distributions of centers-of-mass of TM helices along the membrane lateral directions (x and y) (view from the cytosolic side). The distributions for resting and activated states were derived from the first and last 500 ns of the 750 mV simulation sim 2b, respectively, which were converted into the free energy scale by ∼ R T ln P(x,y) with T = 300 K. The contour for the resting state distribution (dotted lines) is drawn at 4 kcal/mol. d) Overlay of the resting (silver) and activated (orange) states of the TM domain of the Core-MT BK channel. The green and red spheres mark the backbone Cα atom of S4 R213 in the resting and activated states. Only one subunit is shown for clarity but all four filter loops shown for reference.

Electrostatic potential fields of the Core-MT BK channel at 750 mV with resting and activated VSDs.

The fields were calculated as the averages of the first (resting) and last (activated) 250 ns of simulation sim2b. The fields are shown on a plane that goes through the filter and R210. Only two subunits of Core-MT BK are shown for clarity, with side chains of key S4 charges shown in sticks.

Residue contributions to the gating charge per VSD.

Dynamic community, coupling pathways, and information flow of VSD-pore coupling in BK.

a) Dynamic community analysis showing that TM S4-6 are clustered into single tightly coupled community (blue network). The nodes (residues) and edges (contacts) are colored based on the community number. b) Optimal and suboptimal pathways of dynamic coupling between R213 (VSD S4) and E321 (pore-lining S6). All paths are colored green except for the optimal path, which is colored red. Nodes with information flow value > 0.02 is highlighted in purple; c) Information flow profile of the Core-MT BK channel with R213 as the source and E321 as the sink node (labeled by red circle), respectively. All dynamic coupling analysis was derived from last 500 ns of sim 1 (closed state at 0 mV; see Table S1).

Summary of simulations.

The closed state structure is the fully equilibrated conformation derived from the Ca2+-free Cryo-EM structures of the acBK channel (PDB 5tji), and the activated state structure (sim 5 and 6) was taken from the last snapshot of sim2b. The P-loop/filter (T273 to D292) and C-terminus were harmonically restrained with a force constant of 0.5 kcal.mol-1.Å-2 in all production simulations. The 7 VSD charged residues examined in sims 3-6 are D153, R167, D186, R207, R210, R213 and E219.

The charged group z-displacement, z-displacement of the centers of mass of TM helices, number of pore waters, the averaged pore profiles during four Anton 2 simulations of Core-MT BK channels at 0 mV (row 1), 750 mV (rows 2-3) and 300 mV (rows 4-6) membrane voltages. See Methods for additional details of the simulation and analysis.

Distribution of lipid phosphor atoms under different membrane voltages. The distributions were derived from the last 500 ns of simulation in 0 mV (sim 1), 300 mV (sim 9) and 759 mV (sim 2a). Only the phosphate groups not within 12 Å of any protein atoms were selected for analysis. The average distances between phosphor atoms in the upper and lower leaflets are 45.6 Å, 47.0 Å and 47.0 Å at 0 mV, 300 mV and 750 mV, respectively.

Conductance of the open state of Core-MT derived from the Ca2+-bound full-length BK structure (PDB: 5tj6). The voltage was set at 750 mV and ion permeation events are shown as impulses. The conductance estimated from the second half of the trajectory is ∼6 pS.

Steered MD simulations of BK activation.

a) Illustration of the steered MD setup. Initially, the reference point (orange circle) is placed at the position of R210 or R213 guanidinium CZ atom in the resting state (black circle/blue rectangle). From 0-100 ns, the reference point moves along the z-axis (orange arrow) from the resting state position towards the activated state position (red rectangle). A harmonic positional restraint of 5 kcal/mol·Å², applied along the z-axis only, was used between the reference point and the CZ atom, steering the CZ atom to move along the z-axis with the reference. After 100 ns, the reference point remains fixed at the activated state position (red rectangle), allowing the VSD and the rest of the channel to respond to R210 and R213 movements. b and c) Numbers of pore water and S6 tilting angle as a function of simulation time during two of the steer MD simulations that lead to pore opening (black trace: replica 2; red trace: replica 4). d) Overlay of the pore structure at the end of replica 2 (red) and the open Cryo-EM structure (cyan). e) Pore profile at the end of replica 2 (red) in comparison to those from the initial state (dashed line) and the open Cryo-EM structure (black).

Covariance matrices of the Core-MT BK channel before (left) and after (right) voltage-induced activation (sim 2b).

Locking and concerted movements of S4, S5 and S6.

a). The difference of residue-residue contact probability between the closed (sim 1, 0-0.5 μs) and activated state (sim 2b, 9.5-10 μs). b) Side and c) bottom view of the average activated (cyan) and closed (white) structures. The structures are aligned using the filter and P-loop. Dashed lines depict contacts where the probability increased (blue) or decreased (red) by more than 0.4 after activation. The Cα atoms of R207, R210, and R213 are represented as spheres. Residues (K234, L235, E321, and E324) involved in S6 bending are illustrated as yellow and red sticks for closed and activated states, respectively; d) Contact maps of S4-6 before (left) and after (right) voltage activation of Core-MT BK channels.