Abstract
Quaternary ammonium blockers were previously shown to bind in the pore to block both open and closed conformations of large-conductance calcium-activated potassium (BK and MthK) channels. Because blocker entry was assumed through the intracellular entryway (bundle crossing), closed-pore access suggested that the gate was not at the bundle crossing. Structures of closed MthK, a Methanobacterium thermoautotrophicum homolog of BK channels, revealed a tightly constricted intracellular gate, leading us to investigate the membrane-facing fenestrations as alternative pathways for blocker access directly from the membrane. Atomistic free energy simulations showed that intracellular blockers indeed access the pore through the fenestrations, and a mutant channel with narrower fenestrations displayed no closed-state TPeA block at concentrations that blocked the wild-type channel. Apo BK channels display similar fenestrations, suggesting that blockers may use them as access paths into closed channels. Thus, membrane fenestrations represent a non-canonical pathway for selective targeting of specific channel conformations, opening novel ways to selectively drug BK channels.
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Data availability
The maps have been deposited in the Electron Microscopy Data Bank (EMDB) under the following accession codes: blocker-free closed MthK: 9405; TPeA-bound closed MthK: 9406; bbTBA-bound closed MthK: 9407; blocker-free closed A90L MthK: 27459; and TPeA-bound closed A88F MthK: 29605. Atomic coordinates for the three structures have been deposited in the Protein Data Bank (PDB) with accession codes 5BKI, 5BKJ, 5BKK, 8DJB and 8FZ7, respectively. Figure 5 and Extended Data Fig. 8 have raw data associated with them. Raw stopped-flow fluorescence quenching traces are available from the corresponding authors upon reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank L. Yen, M. Kopylov and E. Eng for their support during data collection at the Simons Electron Microscopy Center and the National Resource for Automated Molecular Microscopy, located at the New York Structural Biology Center, which is supported by grants from the Simons Foundation (349247), NYSTAR and the National Institutes of Health (NIH) National Institute of General Medical Sciences (GM103310). We also thank W. Rice and B. Wang for data collection at NYU Langone Health’s Cryo-Electron Microscopy Laboratory (RRID: SCR_019202). The work presented here was sponsored, in part, by NIH GM088352 to C.N., the Australian Research Council (DP210102405 and DP2201035501) to T.W.A. and the National Health and Medical Research Council (APP1141974), the National Computational Initiative (dd7), the LIEF HPC-GPGPU Facility (LE170100200), DE Shaw Anton 2 (PSCA17045P via NIH RC2GM093307) and the Medical Advances Without Animals Trust to T.W.A. and E.F.
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C.F., C.N. and T.A. designed the study. C.F. and S.A. prepared cryo-EM samples and collected and analyzed cryo-EM data. N.S. and C.F. performed mutant screening and stopped-flow assays and analyzed the data. E.F. and T.A. performed and analyzed the MD simulations. C.F., C.N. and T.A. assembled the manuscript and wrote the paper, with input from all authors.
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Extended data
Extended Data Fig. 1 Structures of closed MthK with and without blockers.
Cryo-EM map and atomic model of blocker-free (a), TPeA-bound (b), and bbTBA-bound (c) closed MthK viewed parallel to the membrane. Each subunit is in a different color. The lipids bound to the fenestration were colored red. d. Lipid density in the structures of MthK with TPeA (left) and bbTBA (right). The lipid is in yellow (for carbon atoms) and red (for oxygen atoms) sticks. The lipid density is an overlayed mesh. The pore region of two adjacent MthK subunits shown as red and blue ribbons cartoon. e. Side chains of F87 adopt different conformations in each of the four MthK subunits (red, blue, green, yellow). Density shown as mesh, bbTBA as gold stick and K+ as purple sphere.
Extended Data Fig. 2 Cryo-EM characterization of closed MthK with and without blockers.
Representative micrographs of nanodisc-reconstituted (a) blocker-free, (f) TPeA-bound, (k) bbTBA-bound and (p) blocker-free A90L MthK. Calibration bar is 100 nm. b, g, l, q. Selected 2D class averages. c, h, m, r. Cryo-EM final maps colored by local resolution. d, i, n, s. FSC curves for the gold-standard method in black, and for the atomic model with the cryo-EM map in red. e, j, o, t. Angular distribution of the particles used for the reconstruction.
Extended Data Fig. 3 Cryo-EM data processing workflows.
a. MthK TPeA dataset b. MthK-bbTBA dataset. The TPeA (a) and bbTBA (b) densities are shown separately as mesh, with the atomic models as sticks.
Extended Data Fig. 4 Blocker binding site in MthK and other K+ channels.
a. Sequence alignment of the pore regions of MthK, Human BK and KcsA channels. Residues interacting with blockers (boxed red) are conserved. Identity and homology are indicated as dark and light purple, respectively. b. Modeling of TPeA binding in MthK open state. I84 and F87 side chains are shown as sticks. Note that the side chain of F87 has rotated and no longer contributes to TPeA interaction. c. Modeling of TPeA binding in BK channel. The side chains of L312 and F315 are shown as labeled sticks. d. Overlay of MthK-TPeA and KcsA-TBA (PDB 2JK5) structures. The pore region of two opposing subunits are shown as red and blue ribbons cartoon. TPeA and TBA are shown as sticks.
Extended Data Fig. 5 Membrane fenestrations in BK and other K+ channels.
a. Fenestrations in human BK channel in EDTA (PDB 6V3G), lipid yellow ball and stick. Part of the fenestration was blocked from view by the VSD. b. No fenestration observed in human Ca2+-bound BK channel (PDB 6V38), where fenestration was filled by the bent TM6 helix. c. Left: Small fenestration in closed KcsA (PDB 1K4C). Right: No fenestration in locked-open KcsA (PDB 5VK6). d. No fenestrations in closed (PDB 3SPC) or open (PDB 3SPI) Kir2.2, respectively. e. Left: Size of fenestration (dashed line) in BK with EDTA(PDB 6V3G). Protein rendered in surface representation. The VSDs and part of the S5 helix of the red subunit are removed for clarity. The surrounding residues are labeled sticks. Fenestration is formed by S6 helices from 2 adjacent subunits. Middle: lipid (mesh) binding in BK fenestration. Dashed rectangle indicated the region shown in the right panel, where surrounding residues are labeled sticks.
Extended Data Fig. 6 MD simulation of TPeA binding and membrane partitioning.
a. Attempted unbiased ‘flooding’ snapshot of MD simulation for TPeA+ entering the closed MthK channel (after 4 μs). b. The free energy for TPeA for the first 2 μs (left) and the last 2 μs (right) of the simulation. c. TPeA entering MthK though the fenestration based on 1D US simulation (note the different free energy scales in each panel). The free energy profile for the TPeA molecule is flat in the membrane, as TPeA comes closer to the channel there is a gradual slope and a minimum of −7 kcal/mol at x = 13 Å. The TPeA molecule then encounters a barrier with the discontinuity at x ~ 10 Å, due to lack of sampling, where the TPeA molecule is trying to enter in between the two TM2 helices, motivating the need for enhanced sampling methods in Fig.4. There is a minimum at x < 7 Å where TPeA is inside the MthK pore. Error bars are standard error of means based on 5 blocks (n = 5) following equilibration (see Methods). d. Free energy profile for membrane partitioning using 1D US reveals a free energy minimum of −4.9 ± 0.7 kcal/mol relative to bulk water, extending deep inside the bilayer, reaching to ~12 Å from the center with only ~1 kcal/mol penalty. Simulations used 71 independent simulations, with error bars based on asymmetry of the free energy in left and right leaflets (n = 2) following equilibration (see Methods). Insets show TPeA positioning in the membrane at the free energy well (left) and membrane deformation due to interactions between the TPeA ion and water and lipid head groups when TPeA moves closer to the membrane center (right).
Extended Data Fig. 7 Coordination numbers for TPeA+ ions and lipid-TPeA interactions during entry to the pore.
a. Coordination number for TPeA entering though the gate. As the TPeA molecule enters through the gate the nitrogen dehydrates progressively (blue line). It can also be seen to interact with backbone carbonyl oxygens and carboxylate oxygens (red line). b. Mean number of interactions between the TPeA N and water molecules. When TPeA crosses though the fenestration it is forced to dehydrate, before rehydrating in the pore. c. Mean number of interactions between the TPeA N and protein oxygen atoms. The crossing of the fenestration is helped by interactions between the TPeA N and the protein. d-e describe lipid interactions with TPeA from 1D US simulations for TPeA entering though the gate: d. Mean number of lipid C -TPeA C interactions for each window; and e. number of lipid C -TPeA C interactions over time for TPeA at 1 Å ≤ z ≤ 8 Å. Insets below show typical lipid-TPeA interactions at z = 3 Å. f-g describe lipid interactions with TPeA in 2D US simulations for TPeA entering though the fenestration: f. Mean number of lipid C -TPeA C interactions; and g. number of lipid C -TPeA C interactions over time for windows with TPeA at x = 0 Å and 1 Å ≤ z ≤ 8 Å. Insets below show lipid-TPeA interactions at x = 0 Å and z = 6 Å.
Extended Data Fig. 8 Flux rates and TPeA block in MthK channel mutants.
a. Tl+ flux rates of wild-type, A90L, A88F and V91F MthK after activation by 17.2 mM Ca2+ for 100 ms. Data are mean ± S.D. of 2-3 biological repeats. b. TPeA block dose-response curves of closed (left) and open (right) V91F MthK after 10 s and 100 ms incubation, respectively (IC50closed = 2.05 ± 0.5 μM, IC50open = 4.55 ± 0.1 μM). For comparison, the graphs for MthK WT in dotted lines after the same incubation time are overlayed, from Posson et al. (2015)21. Symbols are mean ± S.D of n = 3 biological repeats. c. Fluorescence quench traces after incubating MthK V91F (in 0 Ca2+) with 3 μM TPeA for 0.1 (black) and 10 (cyan) s and no blocker control (green) (lines include 6 repeats each, see Methods). d. TPeA equilibration plots for closed (left) and open (right) V91F MthK with 3 μM TPeA from data as in c. Solid lines are fits with a first-order exponential decay function, with τclosed = 1.86 ± 0.40 s, and τopen = 83 ± 0.66 ms. The dashed lines are the equilibration plots for 3 μM TPeA with WT MthK, from Posson et al. (2015))21. Symbols are mean ± S.D of n = 3 biological repeats.
Extended Data Fig. 9 Structural features of MthK channel mutants.
a. Cryo-EM structure of A88F MthK closed state displays lateral fenestrations between two TM2 helices from adjacent subunits, rendered as in Fig. 3a to be compared with WT (left) and to highlight fenestration size and the residues that line it (right). The introduced F88 faces towards the TM2 of neighboring subunit and F87 changes orientation so that the fenestration is now closer towards the extracellular side and similar in size to WT. b. Fenestration calculated using the program HOLE indicates a ~3.2 A radius for A88F. Plots of the fenestration cavity radius for WT, A90L, and A88F, as indicated. c. Density (grey mesh) detail of the fenestration area for WT, A90L, and A88F with 2 adjacent subunits (shown in cartoons of different colors). Side chains are indicated and those of L90 and F88 are in yellow sticks for emphasis. d. HOLE plots for the pores of A90L (left) and A88F (right). Only two opposing subunits shown. e. Pore radii as a function of the distance along the pore calculated from d. Selectivity filter (SF), L95, and I99 indicated.
Extended Data Fig. 10 MD simulations of MthK WT, A90L and V91F.
a. Representative structures from MD simulations of WT MthK (left), A90L-MthK (middle) and V91F-MthK (right) with side (top row) and top (bottom row) view. The sidechains of residues 90 and 91 are shown as colored spheres in cyan for unmutated residues, purple for L90 and red for F91. b. An example snapshot of WT MthK with 4 lipid tails (beige spheres) sticking into the fenestration. c. Distribution plots of the fenestration radii for the different channels analyzed with HOLE, as described in Methods. d. Time series of the number of lipid tails in the pore during the simulations for WT MthK (left), A90L-MthK and V91F-MthK (right).
Supplementary information
Supplementary Information
Supplementary Table 1 and Supplementary Figs. 1–5
Supplementary Video 1
Simulation of TPeA entry into the MthK pore through the membrane-facing fenestrations
Source data
Source Data Fig. 5
Statistical source data
Source Data Extended Data Fig. 8
Statistical source data
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Fan, C., Flood, E., Sukomon, N. et al. Calcium-gated potassium channel blockade via membrane-facing fenestrations. Nat Chem Biol 20, 52–61 (2024). https://doi.org/10.1038/s41589-023-01406-2
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DOI: https://doi.org/10.1038/s41589-023-01406-2
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