Aromatic interactions with membrane modulate human BK channel activation

Figures

• 

  Figure 1.. Structure of BK channels and effects of C-linker sequence scrambling on its voltage activation. (a) Key functional domains and structural organization of the human BK channel (hSol1). See main text for definition of domains; (b) Orientation of the pore lining S6 helices and C-linkers in the metal-free (green) and metal-bound (red) states; The dash lines indicate the approximate positions of membrane interfaces. The location of Tyr 332 and Gly 310 are indicated by yellow and blue spheres in the metal-bound and metal-free states, respectively. The black arrow shows S6 movement upon metal binding. (c) Macroscopic currents of WT, K0, K2 and K7 hSlo1 channels. The currents were elicited in 0 [Ca2+]i by voltage pulses from −30 to 250 mV with 20 mV increments for WT and K2 and voltage pulses from −80 to 200 mV with 20 mV increments for K0 and K7. The voltages before and after the pulses were −50 and −80 mV, respectively. (d) Conductance-voltage (G-V) curves for WT, K0, K2 and K7 hSlo1 channels in 0 [Ca2+]i showing significant shifts in the activation voltage (V0.5); All solid lines were fit to the Boltzmann relation (see Materials and methods), with V0.5 of 183.4 ± 3.2 mV for WT; 89.6 ±…

• 

  Figure 1—figure supplement 1.. Structural properties of BK channels. (a) Domain swapping arrangement of BK channels. Shown is the structure of the human BK channels (hSlo1) with chain A colored blue and C-linker colored green. Other domains are made transparent for clarity. (b) The C-linker (green) directly connects the pore lining helix (S6, yellow) to the RCK1 N-lobe (blue) of the CTD. The bound Mg2+ is shown as an orange sphere.

• 

  Figure 1—figure supplement 2.. Packing between C-linker and VSD/RCK1 N-lobe of WT hSlo1. (a) Segments highlighted are: C-linker (residue 329–343, green); RCK1 N-lobe (dark blue, residue 344–427); VSD S0’ (yellow, residue 92–107) and S2-S3 loop (magenta, residue 171–177). The orange sphere marks the position of S337 Cα; Y332 and Y336 are shown as red Licorice. (b) Residue contact map of C-linker (blue labels) with VSD and RCK1 N-lobe (green and black labels) derived from the Cryo-EM structure of the metal-bound state of hSlo1 using the LigPlot server (https://www.ebi.ac.uk/thornton-srv/software/LIGPLOT).

• 

  Figure 1—figure supplement 3.. Comparison of the aSlo1-derived homology model of hBK (orange) with the new Cryo-EM structure (PDB 6V38) (cyan) in the metal-bound state. (a) Overlay of the full-length structures aligned using core residues 100 to 600. The backbone RMSD of two structures 2.18 Å. (b) Overlay of the central pore region (including S6 helices), aligned based on residues 287 to 330. The backbone RMSD of the central pore is 0.87 Å. (c-d) Overlay of the whole TMD structures c) side view, (d) top view, aligned on residues 100 to 343. The backbone RMSD is 2.53 Å. Note that the main difference is in the relative arrangements of VSD and PGD, which is also observed in multiple Cryo-EM structures of the same aSlo1 or hSlo1 constructs and thus likely reflect inherent flexibility of the VSD/PGD packing.

• 

  Figure 1—figure supplement 4.. Structural and dynamic properties of hSlo1 obtained from simulations of the recently published Cryo-EM structures (PDB: 6V3G and 6V38). (a) Root-Mean-Square Fluctuations (RMSF) profiles of residues 230–400 (PGD, C-linker and RCK1 N-lobe). C-linker RMSF is highlighted in the green dashed box and in insert. The Cα RMSD of (b) the whole channel and (c) residues 100–500 (the Core region) as a function of simulation time. RMSF and RMSD were obtained from the 150ns MD simulations with snapshots taken every 0.05 ns.

• 

  Figure 1—figure supplement 5.. A representative structural model of hSlo1 with 12 residues (3x AAG units; colored red) inserted after S337 in the C-linker region (colored green). (a) Side view; (b) top view. For clarity, only the C-linker and S6 helix (yellow) of chain A (colored as blue ribbon) are highlighted.

• 

  Figure 2.. Dynamic properties of BK channels. (a) The C-linker N-C Cα distance during a representative 800-ns MD simulations of the WT hSlo1. (b) Residue Root-Mean-Square Fluctuations (RMSF) profiles of PGD, C-linker and RCK1 N-lobe derived from the same trajectory (green dashed box and shown in the insert). (c-d) Optimal and top 10 suboptimal pathways of dynamic coupling (yellow traces) between D895 in the RCK2 Ca2+ binding site of chain A (Cα colored as pink) and I323 in PGD of chain A (Cα colored as red) in the metal-free (c) and bound (d) states. The C-linker is colored green, S6 of chain A and its neighboring chain (chain C) in blue and grey, respectively. The rest of the channel is shown as transparent ribbons. Note that the pathways in the metal-free state go through the neighboring chain (chain C). Figure 2—source data 1.Data obtained from simulation studies (see Materials and method section structural and dynamic analysis) calculating C-linker N-C Cα distance for both metal-bound and metal-free states of WT hSlo1 channels as depicted in Figure 2a. Figure 2—source data 2.Data obtained from simulation studies (see Materials and method section structural and dynamic analysis)…

• 

  Figure 2—figure supplement 1.. Probabilities of residue-residue contacts between the C-linker and VSD of the neighboring chains. VSD spans residues 45–250, where residues 45 to 53 belong to S0 helix, 92–107 to S0’, 171–177 to the S2-S3 loop, and 225–230 to the S4-S5 loop. The contact probabilities were calculated from atomistic simulations. See Materials and methods for additional details.

• 

  Figure 2—figure supplement 2.. Probabilities of residue–residue contacts between the C-linker and RCK1 N-lobe of the same chain. See Materials and methods for additional details.

• 

  Figure 2—figure supplement 3.. Structural and dynamic properties of the C-linker in WT hSlo1 and mutants. (a) The C-linker N-C distance (329–343 Cα) as a function of time during 800-ns MD simulations. (b) RMSF profiles of residues 230–400 (PGD, C-linker and RCK1 N-lobe) derived from the same MD trajectories, showing limited flexibility of the C-linker (green dashed box and in inserts). (c) Representative snapshots showing the equilibrated conformations of C-linkers in both metal-bound and free states. See Materials and methods for the details of the calculations.

• 

  Figure 2—figure supplement 4.. Structural and dynamic properties of WT hSlo1 and C-linker mutants. The Cα RMSD of (a) the whole channel and (b) residues 100–500 (the Core region) as a function of time during 800-ns MD simulations. (c) Evolution of the number of pore water, showing that all hSlo1 constructs readily undergo dewetting transitions in the metal-free state. See Materials and methods for additional details.

• 

  Figure 2—figure supplement 5.. Optimal (red) and suboptimal (green) dynamic coupling pathways between the critical residues in the Ca2+ and Mg2+ binding sites (Cα colored as purple sphere) and I323 (Cα colored as yellow sphere) in PGD. Whole channel is shown as transparent ribbon with each chain colored differently. S6 and C-linker of chain C are colored in gray and shown with a cartoon representation. (a) Path between R514 (located in RCK1 Ca2+ binding site in CTD; purple sphere) in chain C to I323 (located in S6 helix of PGD; yellow sphere) in chain C. (b) Path from E374 (located in Mg2+ binding site in CTD; purple sphere) in chain C to I323 (located in S6 helix of PGD; yellow sphere) in chain C. (c) Path from D99 (located in Mg2+ binding site in VSD; purple sphere) in chain B to I323 (located in S6 helix of PGD; yellow sphere) in chain C. See Materials and methods for additional details.