A gating lever and molecular logic gate that couple voltage and calcium sensor activation to opening in BK potassium channels

Figures

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  Fig. 1.. Shortening the C-linker increases PO while reducing electromechanical coupling. (A) Apo structure of human BK channel, hSlo1 (6V3G), highlighting different domains. For clarity, domains in the front and back have been removed such that adjacent TMD and CTD domains are from different subunits. For illustration purposes, the C-linker is depicted as a spring connecting the adjacent S6 tail and RCK1, but in reality, this connection occurs within the same subunit (fig. S1). (B) C-linker sequence alignment for mouse, human, and aplysia Slo1 orthologs highlighting location of SYS deletion. (C) Log(PO)-V relations (mean ± SEM) in 0 Ca2+ for ΔSYS, S335A, Y336A, S337A, and WT in 0 and 5.5 μM Ca2+. Solid curves represent HA model fits for WT and ΔSYS. (D) Allosteric coupling factor D (mean ± SD) estimated from HA model fits and qλMAX method as described in the text (open circles are individual estimates) is plotted on a log scale to reflect coupling energy, with dashed lines representing 3 SDs from the WT mean. (E) Mean log(PO)-V relations and fits for ΔSYS, and WT in different Ca2+, with arrows representing the Ca2+-dependent increase in log(PO) at −120 mV, illustrating the reduced…

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  Fig. 2.. Ala scan of S4-S5 linker region. (A) Sequence alignment with secondary structure showing that the S4-S5 linker (highlighted in yellow) is much larger in KV1.2 than in Slo1. Colored text indicates residues mutated in mSlo1. (B) TMD structure of apo hSlo1 highlighting the relationships between the S6 tail (green), S4-S5 linker (blue), and regions mutated at the bottom of S4 (red) and S5 (orange). (C) Differences in V0.5 between Ala mutants and WT mSlo1 in 0 Ca2+ (black) and 70 μM Ca2+ (red). (D) Mean log(PO)-V of F223A and WT in 0 Ca2+. Solid curves are HA model fits. Dashed curve is F223A data superimposed on the foot of the WT curve to highlight the reduction in V-dependent activation. (E and F) Allosteric factor D and basal activity L0 for WT and mutants in 0 Ca2+. For Q222A, I233A, K234A, and L235A, PO was too low in 0 Ca2+ to measure V0.5, D, or L0, but L0 was estimated (red bars) from basal activity in 70 μM Ca2+.

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  Fig. 3.. Electromechanical coupling mechanism in the TMD. (A) Gating lever (green) provides a pathway connecting F223 (S4) and E321 (S6) in the open, Ca2+-bound hSlo1 structure (6V38). (B) Apo closed structure viewed from the cytoplasmic side of the channel shows minimal contact between gating lever residues (purple) and S6. (C) In the Ca2+-bound open structure, gating lever residues K234 and L235 make extensive contact with S6 (E321, I322, L325, and I326), including a salt-bridge interaction between K234 and E321 (dashed line). (D) Changes in S5/S6 interaction and F223 movement are associated with rigid-body rotation of the gating lever and S5 by ~20° (purple: apo closed; green: Ca2+-bound open). Structures were aligned to S5 through the N-terminal end of S6.

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  Fig. 4.. Ala scan of S6 and C-linker. (A) Sequence alignment for KV1.2, MthK, and Slo channels, with conserved hinge residues highlighted, secondary structure indicated for KV1.1 (top) and Slo1 (bottom), and regions mutated indicated by colored bars. (B) Position of hinge residues is indicated in apo hSlo1 structure. (C) Allosteric coupling factor D for mutants in S6 and C-linker compared to WT and ΔSYS, with dashed lines indicating 3 SDs from WT mean. Mean log(PO)-V relations in 0 Ca2+ for (D) WT and hinge mutations (P320A, G333A, G334A, and ΔSYS) or (E) C-linker mutations (R329A, K330A, K331A, and Y332A). Solid curves are HA model fits. Dashed curve is WT mean data in 5.5 μM Ca2+.

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  Fig. 5.. Ala scan for Y332 interaction partners. (A) Allosteric coupling factor D for mutants in Y332 or αB-βC loop of RCK1. Sequence alignment for Slo1 orthologs with mutated positions in green. (B) Mean log(PO)-V relations for F391A, F400A, Y332A, and WT in 0 Ca2+. Solid curves are HA model fits; dashed curve is WT 5.5 μM Ca2+. (C) Structure of αB-βC loop showing proximity of Y332 with potential interaction partners F391 and F400. Apo (tan) and Ca2+-bound (blue) structures are superimposable. Contacts are shown for apo structure. (D) Mean log(PO)-V relations in 0 Ca2+ for WT, Y332A, Y332A/F391A, Y332A/F400A, and F391A/F400A, showing similarity between double mutants and Y332A. Solid curves are HA model fits. (E) Double-mutant cycle analysis showing that interaction of Y332 with both F391 and F400 (but not F391 with F400) contributes to changes in D (blue) and L0 (gray) based on the apparent interaction energy (Ω) determined for these parameters, as described in the text.

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  Fig. 6.. The YFF pathway mediates V- and Ca2+-dependent coupling by suppressing resting activity. (A and B) Plots of basal activity (L0) versus allosteric coupling factor D or C for all constructs tested. Red and blue lines represent inverse fourth and eighth power relations between L0 and coupling factor [e.g., L0(mut) = L0(WT)(DWT/DMut)n, where n = 4 or 8]. Symbols indicate mutation location, S4-S5 linker region (■), S6 (♦), C-linker (▲), or αB-βC loop (▼) and double mutants involving Y332, F391, and F400 (◆). Colors indicate mutants involving Y332, F391, and F400 (red), RKK ring (orange), and G334A or ΔSYS (yellow) and mutants in the TMD that alter coupling (P320, E321A, I322A, and F223A) (blue), with all others being gray. (C) Thermodynamic cycle showing C-O equilibrium constant as it increases from basal level L0 to LV or LCa when all V or Ca2+ sensors are activated or LCa,V when both sensors are activated. ΔG values represent energy change between states. (D) Plot of coupling factors D versus C. Solid line represents equal energetic change in both parameters relative to WT; dashed lines reflect ±SD of the WT value of D. (E) Mean log(PO)-V relations for WT and Y332A. Solid curves are…

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  Fig. 7.. A molecular logic gate mechanism mediated by the YFF pathway. (A) Structures of the αB-βC loop and C-linker from one subunit and VSD from the adjacent interacting subunit, with gating lever residues shown as space filling (green). In the apo closed conformation (top), interaction between R329 in the RKK ring (orange) and E321 in S6 suppresses opening. This interaction is broken when the αB-βC loop undergoes a lateral movement (arrow) upon gating ring expansion in the Ca2+-bound conformation (bottom). (B) Scheme 1 is HA model modified to include a nonadditive component of V- and Ca2+-dependent coupling Y, representing the YFF pathway mechanism. (C) The relationship among allosteric factors and equilibrium constants defined by Scheme 1 is illustrated by a diagram representing the four open and four closed states that can be accessed from rest by activating a single V and/or Ca2+ sensor within a functional unit. (D) Scheme 1 predicts Ca2+-dependent decrease in electromechanical coupling for WT, but not Y332A, consistent with data. (E) Mean GK-V relations for WT in 0, 5.5, and 70 μM Ca2+ are compared to predictions of Scheme 1 (solid curves) at these and additional concentrations…

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  Fig. 8.. Relationship between two electromechanical coupling pathways in Slo1. (A) Gating lever (green), immediately above the S4-S5 linker (cyan), includes S4 and S5 residues that are proposed to interact with the VSD and S6, respectively, in the same subunit (red arrows) in open-activated states to stabilize the open S6. Static interaction of Q222 and I233 at the S4/S5 interface helps maintain gating lever structure as it rotates about the S5 axis during V sensor activation. (B) The YFF pathway, composed of the αB-βC loop in RCK1 (pink) and proximal C-linker (orange), interacts with S6 in an adjacent subunit (red arrow) through the “RKK” ring when V and Ca2+ sensors are at rest to stabilize the closed conformation. However, movement of the αB-βC loop triggered by Ca2+ binding or V sensor activation (blue arrows) disrupts the stabilization of S6, owing to static interaction of Y332 with F391 and F400 at the C-linker/αB-βC loop interface. (C and D) Axial views of the cytoplasmic end of the hSlo1 pore in Ca2+-bound open and apo closed conformations, respectively, with subunits in different colors, showing the contribution of E321 in S6 to different coupling pathways. (C) The gating lever…