A hERG Blocker Facilitates K ⁺ Channel Current by Agonizing Pore Opening while Blocking

Figure 1 –. The pore blocker nifekalant facilitates hERG currents. (A) hERG currents in response to voltage-clamp pulses under control condition after 4s +60 mV prepulse (black), in 100 nM nifekalant with prepulse (blue), in 100 nM nifekalant without prepulse (green). (B) The relationship between the tail current amplitude and test pulse voltage. Means ± SEM (n = 5 cells). Data replotted from Furutani et al., 2019. Prepulse does not change hERG conductance without nifekalant (Furutani et al., 2011).

Figure 2 –. Agonism-while-blocking can produce facilitation. (A) Cartoon depicting the proposed state transitions underlying facilitation. Closed channels (State C, gray) can open (State O, green) and open channels can become blocked (State O*, yellow). Open/blocked channels can close (State C*, red), but State C* is less stable, as indicated by the asymmetric equilibrium arrows. (B) Overlay of the G-V relationship predicted by our simplified kinetic model when [D]=0 and the hERG G-V (reproduced from Furutani et al., 2019). Red curve illustrates the activation-V relationship of blocked channels. (C) Simulated time courses of State O occupancy during a voltage protocol used to elicit facilitation. Inset: curves normalized to the maximal value of the drug-free simulation. (D) G-V curves from peak simulated tail currents with [D]=KD, with (blue) or without (green) a 4 s pre-pulse to +60 mV before each test pulse. G-V curve with [D]=0 shown for comparison. Peak tail open probability normalized by peak tail open probability following +40 mV test pulse with [D]=0. (E) State-to-state flux, probability per second, over time during a facilitated test pulse for unblocking (State O* to State O, dotted…

Figure 3.. Drug docking simulations reveal distinct nifekalant binding poses in open vs. closed hERG channels. (A) closed-state and (B) open-state hERG channel models. Three representative binding poses (Pose 1, 2, and 3) were obtained by clustering the top 50 nifekalant docking results with the most favorable binding free energies from a total of 25,000 poses per hERG channel state. For each pose, the pore radius profiles of the hERG channel with the bound drug are shown alongside the apo model, at the same vertical level as the visualization directly to the left of the plots. The canonical drug binding residues Y652 and F656 on the pore-lining S6 helices are highlighted in gold. 2D interaction maps are shown to illustrate key contacts between nifekalant and the channel, with interactions visualized for two opposing subunits for clarity. (C) Distribution of nifekalant center-of-mass Z-position for the top 50 poses (relative to membrane axis) reveals that the drug significantly shifts upward toward the selectivity filter when it binds to the closed-state model compared to open-state model (Mann – Whitney – Wilcoxon U test, M.W.W., p ≤ 1e−5). Horizontal dashed lines mark the center-of-mass…

Figure 4:. Voltage-dependent affinity suggests that nifekalant prefers activated channels. (A, B) Representative whole-cell current recordings showing hERG channel inhibition during test pulses from a holding potential of −90 mV in control conditions (black traces) and after application of 300 nM nifekalant (red traces). (C) Relative steady-state current remaining (Idrug/Icont) as a function of voltage derived from the experiments shown in panels A and B (n = 6). (D) Upper panel: Representative current traces showing hERG inhibition during test pulses to +40 mV for 4 s, followed by repolarization to −100 mV for 2 s in the control (black) and 300 nM nifekalant (red). Lower panel: Relative steady-state current fit a single-exponential function (red) to tail current ratios (gray) in the presence of 300 nM nifekalant. (E) Voltage-dependence of steady-state fractional block (Idrug/Icont) was quantified from experiments, as shown in panel D (n = 5). (F) Voltage dependence of the time constants (τ) for nifekalant unbinding. (G) Voltage-dependence of nifekalant apparent dissociation constant (KD). KD values were calculated assuming 1:1 binding stoichiometry (open circles: derived from experiments,…

Figure 5:. Facilitation decay is use-dependent. (A) Representative current recordings demonstrating decay of facilitation during repeated test pulses in the presence of 100 nM nifekalant. Facilitation was induced by 4 s prepulses to +60 mV (indicated by red arrows). Following facilitation induction, test pulses of −40 mV (4 s duration) were applied at intervals of 10, 15, or 30 s. (B, C) Time course of the decay of facilitation versus time after prepulse (B) and number of test pulses (C). Facilitation decayed significantly faster at 10 s intervals than at 30 s intervals (10 s vs 15 s, *p < 0.0002; 15 s vs 30 s, *p < 0.0001, 10 s vs 30 s, *p < 0.0001; with one-way ANOVA followed by Tukey's post hoc test). No significant differences in decay time constants were observed when the data were normalized to pulse number rather than time (10 s vs 15 s, *p = 0.9579; 15 s vs 30 s, *p = 0.9752, 10 s vs 30 s, *p = 0.9989; with one-way ANOVA followed by Tukey's post hoc test), indicating that facilitation decay depends primarily on channel activation frequency rather than absolute time. Data are means ± SEM (n = 4).

Figure 6 –. Facilitation in a hERG gating model of agonism-while-blocking. (A) blocked states of hERG (red, with asterisks) were added to the hERG gating model developed by Adeniran et al. (black) with gating modulation implemented via the factors fα and fβ. (B) Simulated percent block (summed occupancies of states: C1*, C2*, C3*, O*, and I*) during a 4 s pre-pulse to +60 mV and subsequent return to holding potential. (C-E) At left, simulated open probability (occupancy of state O) versus time during a −40 mV test-pulse occurring 10 s after a 4 s pre-pulse to +60 mV. Open probability from simulations with [D]=100nM (gray and red traces) are overlayed on traces from drug-free simulations (black). Arrows indicate points used for peak open probability quantification. At right, peak open probability during −60 mV step versus test-pulse voltage. Overlaid are ratios of the two peak open probability-voltage curves (blue, right axis), illustrating the degree of facilitation or block as a function of voltage.

Figure 7 –. Optimization of a hERG facilitation model. Maximum open probability during the −60 mV tail step (following a test-pulse to −40 mV) in simulations. Open probabilities were normalized to control, drug-free simulation. (A) fα and fβ were individually varied. (B) Heatmap of maximum open probability when fα and fβ are both varied. (C) Drug concentration [D] was varied. Optimal facilitator has fα=92 and fβ=10. (D) Drug on and off rates were varied for the optimal facilitator.

Figure 8 –. State transitions underlying facilitation: (A) Simulated hERG state O occupancy (open probability) during a step to −40 mV with a maximally facilitatory blocker: fα=92, fβ=10, [D]=100nM=KD. (B) Occupancy of state I. (C) Summed occupancy of blocked states C1* + C2* + C3* + O* + I*. (D) Summed occupancy of unblocked closed states C1 + C2 + C3. (E) State-to-state flux.