Distinct mechanisms of inhibition of Kv2 potassium channels by tetraethylammonium and RY785
- Theoretical Molecular Biophysics Laboratory, National Heart, Lung and Blood Institute, National Institutes of Health, United States
- Molecular and Cell Biology Graduate Program, Johns Hopkins University, United States
Figures
Figure 1
Structure and simulation of the Kv2.1 channel in the activated state.
(A) Structure of Kv2.1 determined by single-particle cryo-electron microscopy in a lipid nanodisc (Fernández-Mariño et al., 2023), viewed from the cell intracellular side and along an axis perpendicular to the membrane. The channel is an assembly of four identical subunits (in colors); the transmembrane ion pore is formed at the center of the assembly, and the voltage sensors are found in the periphery, in a domain-swapped configuration. A K+ ion is shown in magenta, inside the selectivity filter. (B) Simulation system used in this study, comprising the channel (yellow), a phospholipid bilayer (gray), and a 300 mM KCl buffer (magenta, green). The figure depicts the final configuration of the 25 µs molecular dynamics (MD) trajectory described in Figure 2. The total number of atoms is 201,954, some of which are omitted in the figure, for clarity. (C) Close-up of the pore domain and the selectivity filter. Note two protein subunits are omitted, for clarity. The configuration represented is that shown in panel (B); in this configuration, K+ ions are found in sites S1, S3, and S4 within the selectivity filter, while sites S0, S2, and Scav are transiently vacant.
Figure 2
Mechanism of K+ permeation through the Kv2.1 channel.
(A) Time traces of the position of K+ ions along the central axis of the channel as they reach and permeate the selectivity filter toward the extracellular side (black, red, and blue). Ions that do not reach the filter are not shown for clarity (those that reach the filter but return to the cytoplasmic side before permeating are shown in orange and cyan). The approximate location of each of the K+ binding sites (S0 through S4, and Scav) along the pore is indicated alongside the plot. (B) Same as (A), for a fragment of the trajectory that illustrates the knock-on mechanism that initiates and completes each of the observed permeation events. (C) Density for K+ ions along the channel axis, relative to the bulk concentration value of 300 mM. Density peaks correspond to each of the K+ binding sites within and adjacent to the selectivity filter. For reference, gray horizontal lines indicate the average position of selected protein atoms: in the selectivity filter these are, from top to bottom, the backbone carbonyl oxygens of residues Y376, G375, V374, and T373, and the side chain hydroxyl oxygen of T373; and in the cytoplasmic gate, the alpha-carbon of residue P408. All ions in the simulation system contribute to this profile, but only while they reside in a cylindrical volume of diameter equal to 12 Å, centered in and parallel to the channel axis, extending across the whole system.
Figure 3
Binding of tetraethylammonium (TEA) and RY785 to Kv2.1 channel and impact on K+ permeability.
(A) Time trace of the position of TEA (N atom) in the first 500 ns of the simulation, showing its spontaneous binding to the cavity between the selectivity filter and the cytoplasmic gate (marked by the Cα atoms of T373 and P406, respectively, indicated with gray traces). (B) Time trace of the position of TEA for the rest of the 5 µs simulation, alongside those for K+ ions within the selectivity filter, shown as in Figure 2. The location of each of the K+ binding sites therein is indicated alongside the plot. (C) Close-up of the channel in the final snapshot of the simulation, with TEA bound to Scav. Only two of the four channel subunits are shown, for clarity. Residues P406 and I405 are marked in green. (D–F) Same as (A–C), for the simulation of RY785 binding, indicating separately the positions of the central N atom and of the S atom in the distal five-membered ring.
Figure 4
Bound RY785 does not occlude the K+ pathway, but tetraethylammonium (TEA) does.
(A) Probability histograms for the position of TEA and RY785 (in Å) as projected on the plane of the membrane, i.e., perpendicular to the pore, with the origin on its central axis. (B) Snapshot of the simulation of Kv2.1 and TEA described in Figure 3, with a density map for TEA (blue mesh) calculated for all non-hydrogen atoms and all simulation snapshots. The map is viewed from the membrane plane, on the left, as well as from the cytoplasmic entrance of the pore, on the right. (C) Same as (B), for the simulation of Kv2.1 and RY785. All maps are contoured at the same sigma value (equal to 0.04).
Figure 5 with 1 supplement
RY785 binds to the interior wall in the Kv2.1 cavity.
(A) Close-up of the snapshot shown in Figure 4C, highlighting the most frequently observed protein contacts for RY785; these are on helix S6, which lines the cavity. Only two adjacent protein subunits are shown for clarity. Note that V409, P406, and Ile405 are located at the narrowest point of the permeation pathway (aside from the selectivity filter). For reference, the inset shows an alignment of the sequence of this region of S6 (residues 396–409) with its equivalent in the KcsA K+ channel (residues 98–111), whose closed-state structure is known (Zhou et al., 2001). A more comprehensive comparison of the sequences of the S6 segment in K+ and Ca2+ channels is included in Figure 5—figure supplement 1. (B) Statistical analysis of the interactions depicted in panel (A). A contact was defined as an instance wherein a C atom in the abovementioned hydrophobic side chains was within 4.5 Å of one of the C/S atoms in RY785; a given snapshot might thus show multiple contacts with each side chain, sometimes in two different subunits. The plot in the figure quantifies the percentage of simulation snapshots in which a given number of contacts were observed for each side chain at minimum; e.g., in 80% of the snapshots RY785 forms ~5 or more contacts with Pro406; in 1/4 of those, or 20% of the total, the number of observed contacts is ~10 or more. Contacts with Val409 and Ile405 are mostly in one subunit, while Pro406, Ile401, and Val398 are contacted simultaneously in two subunits.
Figure 5—figure supplement 1
Multiple-sequence alignment of the S6 segment in selected K+ and Ca2+ channels.
(A) Kv2.1 is compared with other Kv channels. (B) Kv2.1 is compared with Cav channels. The degree of sequence similarity is indicated in colors, from dark purple (high) to light purple (low). Orange, green, and cyan rectangles highlight Kv2.1 residues observed to interact with RY785 in our simulations, consistent with the color scheme used in Figure 5. Note that in Cav channels, the S6 sequences differ among the four subunits; thus, each subunit is aligned with Kv2.1 for comparison.
Figure 6
Bound tetraethylammonium (TEA) precludes K+ from accessing the S4 binding site, but bound RY785 allows it.
(A) Five independent simulations of Kv2.1 bound to TEA wherein a knock-on event was artificially induced in the selectivity filter at t = 100 ns, to create a K+ vacancy in site S4. K+ did not reload this site in the subsequent 100 ns, except when TEA spontaneously dissociated prior to the knock-on event (right). (B) Same as (A), for Kv2.1 bound to RY785. In all simulations, a K+ ion reloads the S4 site within ~10 ns of the induced knock-on event, corroborating RY785 is not an open-state blocker of Kv2.1.
Figure 7
Hypothetical mode of Kv2 channel inhibition by RY785.
R785 does not directly block K+ flow; instead, it reshapes the conformational free-energy landscape of the pore domain to stabilize an occluded, partially closed state that does not require S4 to reach the downstate in full. The model is an inference based on the simulation data reported in this article and a previous electrophysiological study by Marquis and Sack, 2022. Note that this is a simplified diagram of the functional cycle of the channel, which includes nonconductive or transiently inactivated states, aside from the fully deactivated form (Stix et al., 2023; Tan et al., 2022).
Appendix 1—figure 1
Chemical structures of (top) RY785 and (bottom) tetraethylammonium.
Atom names and partial electronic charges are indicated.
Appendix 1—figure 2
Potential-energy curve for the C46-C42-N43 bond angle in RY785 between 100° and 135°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 3
Potential-energy curve for the C42-C39-N38 bond angle in RY785 between 100° and 145°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 4
Potential-energy curve for the C39-C42-N43 bond angle in RY785 between 95° and 140°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 5
Potential-energy curve for C42-C39-N40 bond angle in RY785 between 105° and 150°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 6
Potential-energy curve for the N40-C39-N38 bond angle in RY785 between 95° and 135°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 7
Potential-energy curve for the C25-N27-C29-C30 dihedral angle in RY785 between 0° and 180°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 8
Potential-energy curve for the N40-C39-C42-N43 dihedral angle in RY785 between –200° and 0°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 9
Potential-energy curve for the C39-C42-N43-C44 dihedral angle in RY785 between 120° and 240°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 10
Potential-energy curve for the C46-C42-N43-C44 dihedral angle in RY785 between –60° and 60°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 11
Potential-energy curve for the C42-C39-N40-C33 dihedral angle in RY785 between –240° and –120°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 12
Potential-energy curve for the N38-C39-N40-C33 dihedral angle in RY785 between –60° and 60°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 13
Potential-energy curve for the C42-C39-N38-C32 dihedral angle in RY785 between 120° and 240°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 14
Potential-energy curve for the C42-C39-N38-H41 dihedral angle in RY785 between –60° and 60°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 15
Potential-energy curve for the N40-C39-N38-C32 dihedral angle in RY785 between –60° and 60°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 16
Potential-energy curve for the N40-C39-N38-H41 dihedral angle in RY785 between –240° and –120°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 17
Potential-energy curve for the S45-C46-C42-C39 dihedral angle in RY785 between –240° and –120°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray) by scanning.
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 18
Potential-energy curves for the S45-C46-C42-N43 dihedral angle in RY785 between –60° and 60°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 19
Potential-energy curve for the H48-C46-C42-N43 dihedral angle in RY785 between 120° and 240°, calculated with MP2/6-31G(d) (blue) and with default CGenFF (orange) or with our optimized force field (gray).
The Y axis shows the energy (in kcal/mol) relative to the lowest-energy conformer.
Appendix 1—figure 20
Ab initio optimized geometries of RY785-water complexes used in the calibration of our molecular-mechanics force field for RY785.
Appendix 1—figure 21
Interaction energies (in kcal/mol) for the RY785-water complexes shown in Appendix 1—figure 20.
QM data (Y-axis) are correlated with MM values calculated using the default CGenFF (red) and our optimized force field (black). The QM interaction energies are calculated with HF/6-31G(d) and scaled by a factor of 1.16. The dashed line represents the equation QM interaction energy = MM interaction energy. The default CGenFF for RY785 results in an average unsigned error of 1.2 kcal/mol as compared to an average unsigned error of 0.3 kcal/mol for the optimized model.
Tables
Appendix 1—table 1
Interaction distances (in Å) and interaction energies (in kcal/mol) for the RY785-water complexes shown in Appendix 1—figure 20.
QM data are compared with MM values calculated using the default CGenFF and our optimized force field.
| Complex | EQM | rQM | EMM, def | rMM, def | EMM, opt | rMM, opt |
|---|---|---|---|---|---|---|
| A | –1.83 | 2.58 | –1.76 | 2.60 | –1.86 | 2.59 |
| B | –2.12 | 2.51 | –1.82 | 2.60 | –1.90 | 2.60 |
| C | –1.71 | 2.61 | –3.56 | 2.53 | –2.07 | 2.64 |
| D | –2.34 | 2.47 | –3.83 | 2.51 | –2.32 | 2.62 |
| E | –4.92 | 1.99 | –6.70 | 1.83 | –4.22 | 1.92 |
| F | –3.35 | 2.14 | –5.53 | 1.87 | –3.88 | 1.94 |
| G | –8.17 | 2.06 | –7.15 | 1.88 | –7.89 | 1.87 |
| H | –9.44 | 2.04 | –7.11 | 1.88 | –7.24 | 1.88 |
| I | –5.56 | 2.09 | –7.75 | 1.85 | –3.90 | 1.97 |
| J | –3.84 | 2.07 | –9.76 | 1.83 | –4.75 | 1.95 |
| K | –3.81 | 2.32 | –3.38 | 2.23 | –3.84 | 2.22 |
| L | –3.93 | 2.30 | –3.34 | 2.24 | –3.82 | 2.22 |
| M | –2.06 | 2.31 | –1.86 | 2.26 | –2.04 | 2.25 |
| N | –2.81 | 2.22 | –2.20 | 2.22 | –2.51 | 2.21 |
| O | –0.67 | 2.86 | –0.38 | 2.60 | –0.40 | 2.62 |
| P | –0.59 | 2.92 | –0.49 | 2.59 | –0.48 | 2.60 |
| Q | –0.40 | 2.94 | –0.12 | 2.66 | –0.29 | 2.67 |
| R | –0.82 | 2.80 | –0.29 | 2.58 | –0.37 | 2.57 |
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Distinct mechanisms of inhibition of Kv2 potassium channels by tetraethylammonium and RY785
eLife 13:RP101855.
https://doi.org/10.7554/eLife.101855.4
