Pore-modulating toxins exploit inherent slow inactivation to block K⁺ channels

Significance

The toxic arsenals of venomous organisms contain polypeptide toxins that block ion channels to enable the rapid paralysis of a potential prey or foe. In potassium channels, the open conformation relies on the spatial distribution of structural water molecules buried in protein pockets that surround the central pore. Here, we describe a peptide isolated from a cone snail that blocks voltage-gated potassium channels by disrupting the intramolecular hydrogen bond network that governs the dynamic distribution of these structural water molecules. This insight exposes the peripheral water pockets of ion channels as novel pharmacological targets and provides cues for the design of therapeutics active at these sites.

Abstract

Voltage-dependent potassium channels (K_vs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K⁺-selective pore. Animal toxins targeting K_vs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K⁺ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of K_v1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K⁺ channels as a novel pharmacological target and provides a rational framework for drug design.

Voltage-gated potassium channels (K_vs) shape the electrical properties of excitable cells by facilitating the selective flow of K⁺ ions across the cell membrane in response to changes in membrane potential. The K_v channel protein is composed of a pore domain that enables ion translocation, surrounded by 4 voltage-sensing domains that gate the pore in a voltage-dependent manner (1). The selectivity for K⁺ is contributed by a 5-residue signature sequence (TVGYG) termed the selectivity filter (SF) that forms a constriction of the pore at the extracellular face of the channel (2). To pass through the SF, K⁺ ions must shed their hydration shells. Having their backbone carbonyl oxygen atoms facing the pore and creating 4 consecutive coordination sites (s1 to s4) for a dehydrated K⁺ ion, the signature residues catalyze this reaction (3). At the SF, the alternate binding of K⁺ ions and water molecules in a single file enables a “knock-on” mechanism, wherein an incoming ion exerts electrostatic repulsion that displaces a neighboring ion along the pore, resulting in conduction (4, 5).

In addition to its role in setting the preference for K⁺ ions, the SF serves as a gate that down-regulates ion flow via a process known as C-type or slow inactivation. Slow inactivation is triggered during prolonged depolarizations, and is antagonized by ion occupancy in the pore (6–8). While it is widely accepted that slow inactivation entails a conformational change that renders the SF nonconductive, the exact molecular description of this state is still under debate. The crystal structure of the bacterial channel KcsA at low external K⁺ has revealed a “pinched” SF in which the protein backbone was bent at G77 and ion coordination sites s2 and s3 were unoccupied (3). This channel conformation was suggested to be sterically stabilized by a network of water molecules buried in cavities behind the SF (“peripheral cavities” hereafter; ref. 9). Residues situated near these cavities that serve as hydrogen bond donors/acceptors were proposed to shape the rate of interconversion between the open and the inactivated channel states (10). This proposition is supported by the recent structure of the K_v1.2-2.1 paddle chimera in lipid nanodiscs in which these hydrogen bonds are compromised (11).

Many venomous organisms carry in their arsenal peptide toxins that block K_v channels, thus lowering the threshold for an action potential at the affected nerve or muscle, leading to paralysis. These toxins have traditionally been classified into 2 groups according to their mode of action (12). Gating modifier toxins block the channel by binding its voltage-sensing domain, thereby altering the stability of the closed state. Pore-blocker toxins bind at the pore module and physically occlude the ion permeation pathway. Classical K_v pore blockers from scorpion venom are short polypeptides, typically 30 to 40 residues long, with a rigid core composed of an alpha helix and an antiparallel 3-stranded beta sheet. Early experiments, in which the interactions of the scorpion neurotoxins Charybdotoxin (CTX) and Agitoxin2 with K_vs were probed, revealed that a bound toxin can be dissociated by K⁺ ions entering the channel from the cytoplasmic side, a phenomenon termed trans-enhanced dissociation (13). The latter phenomenon was completely abolished on neutralization of the highly conserved K27 toxin residue (14, 15). A model in which the K27 ε-amino moiety of the bound toxin competes with K⁺ ions on a binding site at the extracellular face of the conduction pathway was put forward (16). This model was validated when the crystal structure of CTX in complex with a mammalian K_v channel was solved, revealing that the toxin projects the sidechain of K27 into the channel pore, and as a result, K⁺ binding at the s1 site is lost (17).

In addition to these pore-blocking toxins, numerous studies have illuminated another diverse family of toxins that target the pore domain of K⁺ channels and block the ionic current without physically occluding the pore. Instead, these toxins exhibit a pharmacophore made of a ring of positively charged residues, and their binding sites are confined primarily to the channel turrets (18–22). The precise molecular mechanisms by which toxins of this family block their targets remain obscure, although a general concept (turret-block) whereby the toxin acts as a lid above the pore entry was proposed (18, 22).

Here we describe a block mechanism used by Conkunitzin-S1 (Cs1), a 60-residue peptide toxin isolated from Conus Striatus, which binds to the drosophila Shaker turrets. We show that instead of directly plugging the ion conduction pathway, Cs1 modifies the permeation of water molecules into the peripheral cavities, thus creating highly asymmetric water distributions around the SF that trigger a local collapse of the channel pore, analogous to slow inactivation. In addition to the description of the pore-modulatory action of the toxin, the data provide general insights into the usage of the peripheral cavities of potassium channels as an important therapeutic target and a framework for rational drug design to affect channel function.

Results

Molecular Dissection of the Cs1–Shaker_KD Complex.

Cs1 is a 60-residue toxin isolated from the venom of the fish-hunting cone snail Conus striatus (23). The toxin molecule has a conserved Kunitz domain fold composed of an N-terminal 3-10 helix, 2-stranded beta sheet, and C-terminal alpha helix reticulated by 2 disulfide bridges (24). Our initial analysis of the toxin has indicated that it is highly toxic to fish larvae (Danio rario, LD₅₀ = 500 nM in bath application), practically nontoxic to mice (LD₅₀ > 50 μg/g by s.c. injection), and has a considerable preference for insect over mammalian K_v channel isoforms (SI Appendix, Fig. S1A; see also ref. 25). While the mammalian K_v isoforms we examined were only partially blocked by 5 μM Cs1, the toxin had moderate affinity for the drosophila shaker channel (dmK_v1.2; EC₅₀ = 234 ± 37 nM; SI Appendix, Fig. S1A) and a high affinity for its K427D mutant (Shaker_KD hereafter; EC₅₀ = 0.95 ± 0.2 nM). Aimed to decipher the structural basis for the apparent preference of Cs1 for insect K_vs, we carried a molecular dissection of the toxin-channel complex. First, the solvent-exposed residues of Cs1 were substituted by alanine, the resulting toxin mutants were expressed and purified, and their potency on the high-affinity K427D mutant of the drosophila shaker channel expressed in Xenopus oocytes was assayed (Fig. 1 A and B and SI Appendix, S1 B–E and Table S1). This analysis has pinpointed 5 toxin residues important for toxicity: R49, Y51, R55, and Y59 at the c-terminal α-helix of the molecule and R34 at the second β-strand. Site-directed mutagenesis targeted to the extracellular residues of the channel pore domain revealed 3 residues with critical contributions to Cs1 binding: the aromatic F425 and the nearby negatively charged D427 and D431 (Fig. 1 C and D and SI Appendix, Fig. S1 F and G). We concluded the molecular dissection with a double-mutant cycle analysis (15, 26) to expose putative toxin-channel amino acid pairwise interactions (Fig. 1E and SI Appendix, Fig. S1H). This analysis revealed substantial coupling energies between R34 of the toxin and all 3-channel residues, as well as between F425 of the channel and R49 and Y59 of the toxin. We set out to obtain a putative structure of the channel–toxin complex by employing a complementary, independent approach of nonconstrained rigid-body docking. Toward this aim, we crystalized and determined the structures of the Q54A and R55D toxin mutants at 1.3 Å resolution (SI Appendix, Fig. S1E), from which we obtained a high-quality model of the wild-type toxin. The toxin molecule was docked onto a channel model that was based on the 2.4-Å crystal structure of the closely related k_v1.2-k_v2.1 paddle chimera (27). Clustering the obtained docked structures, we observed a dominant toxin binding mode that accounted for 836 of the top 1,000 solutions. This model was highly compatible with the distance restraints provided by the experimental coupling energies, and it was stable during prolonged molecular dynamics (MD) simulations (>0.4 μs). The quality of the docked Cs1 model after MD refinement was assessed using the Molprobity structure-validation web service (28). This assessment indicated that the model exhibits favorable stereo-chemical properties and ranks highly compared with other complex structures deduced from experimental restraints (Dataset S7 and ref. 29). The Single Amino Acid Mutation related change of Binding Energy (SAAMBE) method (30) was employed to assess the correlation of the docked model with the experimental results (Fig. 1F and SI Appendix, Table S1). We observed a strong correlation (R² = 0.78) between the experimentally determined changes in the binding affinity of 24 toxin mutants to the predicted values computed on the basis of the docked model. A notable exception was the critical Y59A substitution, whose effect was underestimated by the SAAMBE scoring functions (0.67 kcal/mol predicted vs 4.03 kcal/mol measured). Overall, these analyses suggest that the docked model is highly compatible with the available experimental restraints.

Fig. 1.

Shaker_KD–Cs1 complex: molecular dissection. (A) Dose–response curves of Cs1 high-impact mutants on Shaker_KD expressed in Xenopus oocytes. Each curve was determined in 3 independent experiments or more; error bars indicate SD. The solid lines are best fits to a Hill equation with the Hill coefficient restricted to 1. The EC₅₀ values obtained were 0.95 ± 0.03 nM (WT), 42 ± 6 nM (R34A), 21 ± 5 nM (R49A), 12 ± 3 nM (Y51A), 11 ± 1.4 nM (R55A), and 995 ± 37 nM (Y59A). Data supporting the structural integrity of the resulting toxin mutants are presented in SI Appendix, Fig. S1 C and E. (B) The bioactive residues of Cs1. High-impact toxin residues identified by directed mutagenesis are rendered in sticks and colored to match A. (C) Dose–response curves of Cs1 on the 3 high-impact channel mutants. The extracted EC₅₀ values were 0.95 ± 0.03 nM (WT), 15.2 ± 2.7 nM (D431A), 219 ± 37 nM (D427K), and 693 ± 18 nM (F425A). (D) Top view of Shaker_KD pore domain; channel residues critical for Cs1 binding are rendered in space fill. (E) Double-mutant cycle analysis of Shaker_KD–Cs1 interactions. The absolute ΔΔG values obtained by assaying the indicated toxin mutants on the indicated channel mutants are shown. A 1 kcal/mol threshold was applied to isolate the high-impact interactions. (F) Correlation diagram between changes in free binding affinity calculated from the measured effect of 24-point mutations in Cs1 to those predicted by the SAAMBE method (ref. 30; see SI Appendix, Table S1 for the raw data). (G and H) MD-equilibrated structure of the Shaker_KD–Cs1 complex, top (C) and side (D) views. Channel and toxin molecules are rendered in blue and red, respectively. Channel residues are indicated using a chain:residue notation.

The model revealed a network of hydrogen bonds and salt bridges between the side chains of Shaker_KD:D447 from 3 of the 4 channel domains and the C-terminal residues of the toxin (Fig. 1 G and H). These interactions could not be deduced by molecular dissection, since substitutions at Shaker_KD:D447, which resides at the SF entrance, result in nonconductive channel mutants (31). The positively charged side chain of Cs1:R34 interacts with a negative binding pocket formed by Shaker_KD:D427 and D431, rationalizing the weak binding of the toxin to the wild-type channel bearing a lysine at position 427 (SI Appendix, Fig. S1A). The structural basis for the preference of Cs1 for insect over mammalian K_v channels can be readily inferred: the side chain of F425 from channel subunits B, C, and D has high-impact interactions with Y59, R49, and R34 of Cs1, respectively. The high diversity of these interactions (π–π, cation–π, and aromatic–aliphatic stacking interactions) mandates an aromatic residue at this position for high-affinity binding. This criterion is frequently met by K_v1.2 isoforms isolated from crustaceans, fish, and insects, but it is not observed in mammals, consistent with the preferential toxin action on the natural prey of the cone snail (SI Appendix, Fig. S1K).

Cs1 Does Not Directly Block the Ion Conduction Pathway.

A distinctive feature of the modeled Cs1–Shaker_KD complex that became evident during early MD refinements was that the toxin does not physically occlude the channel pore. Cs1 is bound slightly off the central pore axis, and none of the toxin residues interacts with the SF backbone carbonyls, as do classical K_v pore blockers (16). In MD simulations conducted with bound Cs1, water exchange between the outer vestibule of the channel pore and the bulk was observed, albeit at reduced rate compared with the unmodified channel (Fig. 2 A and B). The gap between the toxin molecule and the channel pore was often occupied by a fully hydrated K⁺ ion (Movie S1). In stark contrast, MD simulations of the classical pore blockers CTX and ShK (32) bound to their respective receptors, as well as the CTX K27M mutant, for which a crystal structure is available (17), revealed a tight seal formed by the toxins around the channel pore (Fig. 2 A and B and Movie S1). This disparity between the simulated behavior of Cs1 and the canonical pore blockers can be experimentally tested using trans-enhanced dissociation assays (13). In these experiments, we assayed toxin dissociation during strong depolarization, which drives K⁺ ions to compete with a bound toxin on the s1 ion coordination site.

Fig. 2.

Cs1 alters, but does not block, water access to the extracellular pore entry. (A) 200 ns MD trajectories of Shaker_KD alone (control) or in complex with Cs1/Shk and of the K_v1.2-2.1 paddle chimera complex with CTX or the CTX K27M mutant, were segmented into 20 × 10 ns windows. At each segment, the number of water molecules passing through a cylindrical volume positioned just above the SF (depicted in B, CTX) were counted (unmodified, 95.6 ± 26; Cs1, 53.95 ± 21.88; ShK, 2.2 ± 2.9; CTX, 0; and CTX K27M, 0.51 ± 0.68 unique water molecules per segment). (B) Cross sections through the protein channel complex, taken at the end of a 20-ns equilibration MD run, revealing the water/ion occupancy at the vicinity of the pore. Two opposite channel subunits (gray) bound to a toxin molecule (cyan) are depicted. The bioactive residues at the C terminus of Cs1, as well as K22 and K27, of Shk1 and CTX, respectively, are shown. K⁺ ions and water molecules residing within the pore or at up to ±5 Å vertical displacement from it are rendered in space-fill. A cylinder of 2 Å radius and 3 Å in height positioned 1 Å above the plane of the outermost SF residue (G375), used for the water traffic measurements in A, is overlayed on the CTX complex structure. (C) Cs1 is insensitive to transenhanced dissociation. Toxins were applied at a dose inducing ∼80% current block, and dissociation was induced using the voltage protocol depicted at the Inset. The effect of CTX (5 μM), AgTx1 (5 nM), and ShK (2 nM) was completely reversed upon strong depolarization with a single exponential time-course (τ = 3.0 ± 0.69 ms [n = 4]; 51 ± 14 ms [n = 7] and 405 ± 51 ms [n = 4] for CTX, AgTx1, and ShK, respectively). Conversely, Cs1 remained bound throughout the experiment (P2/P1 ∼ 1). (D) Typical currents recorded during P2 as a function of Δt in the presence of Cs1 (red) or ShK (black). P2 Currents from individual traces are overlaid using an arbitrary horizontal displacement, Δt is the length of the depolarizing pulse preceding P2.

While the canonic pore blockers CTX, AgTx1, and ShK could be readily knocked off their binding sites by prolonged depolarization, Cs1 remained bound to the channel throughout these experiments, consistent with the proposed docking model in which none of the Cs1 residues is bound at the s1 site (Fig. 2 C and D and SI Appendix, Fig. S2 A and B). This conclusion is further supported by the toxin dissociation curves shown in SI Appendix, Fig. S2C, which demonstrates that Cs1 is resilient to trans-enhanced dissociation, despite having a relatively fast dissociation rate on washout.

Cs1 Induces an Asymmetric Constriction of the Selectivity Filter.

The results presented thus far strongly negate a physical block of the channel pore by Cs1 and suggest that water molecules and ions from the extracellular milieu may access the outer channel vestibule in the presence of a bound toxin. Yet ion flow is blocked completely when a saturating toxin concentration is applied (SI Appendix, Fig. S1A). To resolve this apparent conundrum, we simulated the behavior of the toxin-bound channel during a series of 200-ns time windows. In 5 of 7 simulations carried, we observed a nonsymmetric collapse of the channel pore at the SF region (Fig. 3 and SI Appendix, Table S3, trajectories 1 to 7). The observed collapse occurred typically after a few tens of nanoseconds into the production run and followed a common pattern of molecular events, depicted in Fig. 3. In all simulations, the SF was initially found at 2,4 ionic configuration, with a K⁺ ion bound at s2 and additional ion fluctuating between s4 and the intracellular cavity of the channel (Fig. 3B). At this stable ionic configuration (in the absence of an electric field), the SF assumed a symmetric conformation with a cross-subunit distance of ∼8 Å (Fig. 3C). The initial trigger for pore collapse was the flip of the backbone carbonyl at G444 or Y445 away from the pore into the peripheral cavity (Fig. 3A). This event was closely followed by the dissociation of the K⁺ ion bound at s2, leaving the SF with a single ion occupying s4, and water molecules at the remaining sites (Fig. 3B). This configuration of the SF was highly unstable and triggered a rapid asymmetric collapse of the channel pore, in which a single pair of opposing subunits assumed a pinched conformation typified by a 5.5-Å cross-subunit distance (Fig. 3C). This phenomenon was unique to Cs1 simulations and was not observed in control simulations using either unmodified channels (Fig. 3 and SI Appendix, Table S3, trajectories 8 to 10), obtained using the very same starting configuration with the toxin molecule removed, or complexes of the classical pore blockers, CTX and ShK, bound to K_v channels (SI Appendix, Fig. S3, trajectories 11 to 14). On the contrary, in the latter cases, the constant occupancy of ion coordination site s1 by the ε-amino group of the conserved toxin lysine stabilized the SF conformation, manifested in decreased fluctuations of the backbone atoms and the bound ions (SI Appendix, Fig. S3).

Fig. 3.

Cs1 induces an asymmetric constriction of the selectivity filter. The integrity of the selectivity filter region during 200-ns-long trajectories of unmodified (Left, trajectory #8 SI Appendix, Table S3) and CS1-bound (Right, trj #1) Shaker_KD channels. Drawings to the right of each time series depict the monitored parameters at the 200-ns point. (A) Flipping of the backbone carbonyl atom of G444(GYG). The radius of gyration (ρ) is a measure of the distance between the oxygen atom and the geometrical center of the pore. In the unmodified channel ρ is kept at 2.41 ± 0.19 Å throughout the simulation; in the toxin-bound channel, it has increased from 2.62 ± 0.08 Å (0−50 ns) to 4.8 ± 0.11 Å (150−200 ns). (B) The vertical coordinates (Z) of K⁺ ions in the SF along the MD trajectory. Alternating red-blue stripes at the background delineate the s1 to s4 ion coordination sites. The drawing depicts the SF region of 2 diagonally opposed subunits. Ions are rendered as solid spheres and colored to match the time-series plot. Pore-resident water molecules are rendered in CPK. Three Ions remain bound to the SF of the unmodified channel throughout the trajectory, whereas an ion originally coordinated at s2 is lost in the toxin-bound channel at t ∼ 100 ns. (C) Cross-subunit distance between the Cα atoms of G444 residues from chains A and C (red) or B and D (black). This distance is kept at 8.3 ± 0.14 Å during the unmodified channel simulation. During the toxin-bound simulation, an asymmetric constriction of the pore is observed after the dissociation of the ion from s2 (b), as GG (chain B: chain D) is decreased from 8.59 ± 0.24 Å (0 to 50 ns) to 5.8 ± 0.6 Å (150 to 200 ns), while GG (chain A: Chain C) remains roughly unchanged. The drawing provides a top view of the channel pore, the Cα atoms of G444 residues are rendered in space-fill, and the GG distances are indicated.

Cs1 Modifies Water Permeation into the Peripheral Cavities.

The observed collapse of the channel pore in the presence of Cs1 was initiated by a flip of a backbone carbonyl at G444 (Fig. 3A). This pore residue is positioned ∼11 Å away from the nearest toxin residue (R55) and ∼15 Å away from the channel turrets, where all high-impact interactions with the toxin take place. How can Cs1 remotely trigger a cascade of events that leads to the collapse of the pore? A long-range allosteric effect mediated by the turret residues, which contribute most of the Cs1 binding site, seemed unlikely since substitutions at these residues had no apparent effect on the stability of the open state (33), and comparison of the conformational dynamics of the channel backbone during system equilibration phase with and without bound toxin did not reveal any signs for induced fit [backbone rmsd between the initial and the equilibrated structures was 1.9 ± 0.07 Å and 1.93 ± 0.16 Å (n = 5) for toxin-free and toxin-bound simulations, respectively]. We therefore focused on the interactions between the bound toxin and channel residues in the vicinity of the pore region. The central pore of K⁺ channels is surrounded by water-filled cavities, which were proposed to shape the rate and extent of the slow inactivation process (33). In K_v1.2, the confinement of water molecules within these peripheral cavities is achieved by 2 sets of channel residues. The aromatic cuff (SI Appendix, Fig. S4A and ref. 9), composed of the side chains of Y445, W434, and W435 alongside V443, forms a hydrophobic barrier at the base of the cavity (lower barrier). At the top of the cavity, the side chains of D447, M448, and W434 from one channel subunit and T449 from a neighboring subunit form a barrier that limits the exchange of water molecules between the cavity interior and the extracellular bulk (upper barrier; SI Appendix, Fig. S4B and Movie S2). In simulations of toxin-free channels, the space between the 2 barriers is stably populated by 2 to 3 water molecules that exchange with the bulk at a typical average rate of ∼30 molecules per 100 ns (SI Appendix, Fig. S4G). Since this exchange involves the crossing of the upper barrier, its rate depends on barrier dynamics. In particular, we find high correlation between the rate of exchange of cavity water and the dynamics of the hydrogen bond formed between the side chains of D447 and W434 (SI Appendix, Fig. S4G), which was previously dubbed a molecular timer that sets the pace of slow inactivation (10). Since we find a strong correlation between its integrity to the rate of water exchange via the upper barrier, we refer to it hereafter as the D447:W434 gate, or simply the D-W gate. Analysis of Shaker_KD trajectories in the presence of Cs1 reveals a major impact of the toxin on the dynamic behavior of the D-W gate. D447, positioned directly above the selectivity filter, interacts electrostatically with toxin residues in 3 of the 4 channel subunits (Figs. 1 and 4A and SI Appendix, Fig. S4D). These contacts allow the toxin to modify the D-W gate in a nonsymmetrical fashion. In channel subunit C, a hydrogen bond and a salt bridge between D447 to Y51 and R55 of the toxin, respectively, stabilize the D447:W434 hydrogen bond, leading to a closed conformation of the D-W gate (Fig. 4B). Conversely, in subunit D of the channel, a salt bridge between Cs1:R49 and D447 pulls the later away from W434, leading to a constantly open gate conformation (Fig. 4B and SI Appendix, Fig. S4H). In addition, we observed strong effects exerted by the interactions of Cs1:Y59 with the upper barrier residues in channel subunit B (SI Appendix, Fig. S4D) on the permeation of water into the peripheral cavity.

Fig. 4.

Cs1 modifies water permeation into the peripheral cavities. (A) Ribbon diagram depicting the key interactions between the alpha helical segment of the toxin (tan) and the D447 sidechains of subunits C (cyan) and D (gold). Green labels indicate distances in angstroms. (B) Conformational dynamics of the W434-D447 bond in subunits C (cyan) and D (gold) of unmodified and toxin-bound Shaker_KD. The distance between the O_δ1/O_δ2 atoms of D447 and the N_ε1 atom of W434 along a 200 ns trajectory is plotted, a broken line marks the cutoff distance for a hydrogen-bond. (C) The root-mean-square fluctuations (RMSF) of water molecules inside the peripheral cavities of unmodified (SI Appendix, Table S3, trajectories 8 to 10, 17, 18) and toxin-bound (SI Appendix, Table S3, trajectories 1 to 5) Shaker_KD. Each data point represents the mean RMSF over an independent 200-ns production run. (D) Spatial distribution of water molecules within the peripheral cavities of subunits C (cyan) and D (gold) over a 200-ns trajectory. Dots mark the centers of the water oxygen atoms. The sidechain of V438 positioned at the base of the aromatic cuff barrier is rendered in spheres.

Asymmetric Water Permeation into the Peripheral Cavities Promotes Pore Collapse.

The asymmetric effect of Cs1 on the open probability of the D-W gate in discrete channel subunits directly translated in MD simulations into an asymmetric rate of water exchange through the upper barriers. In toxin-free simulations, the rate of water exchange between the peripheral cavities and the bulk solvent through the upper cavity barriers was 31.75 ± 2.4 molecules per 100 ns, evenly distributed among the 4 channel subunits (SI Appendix, Fig. S4E). In Cs1-bound simulations, the rate of water exchange through the upper barrier in subunit D, which had a permanently open D-W gate, was nearly 4-fold higher compared with in subunits B and C, which had their D-W gates in predominantly closed conformations (SI Appendix, Fig. S4 E and H). This resulted in a high thermal agitation of water molecules within the peripheral cavity of subunit D compared with the other subunits (Fig. 4C). These highly disordered water molecules could not be stably confined within the peripheral cavity boundaries; instead, they spread to neighboring channel regions by triggering a flip of the aromatic ring of Y445, thereby breaching the hydrophobic barrier at the bottom of the cavity (Fig. 4D, Movie S3, and SI Appendix, Fig. S4). The breach of the lower barrier allowed the exchange of water with the neighboring cavities (Movie S3 and SI Appendix, Fig. S4H) or with the intracellular vestibule through a path contributed by the hydrophilic side chains underneath (SI Appendix, Fig. S4F). In both routs, the flow of water behind the selectivity filter could be diverted into the central pore by a flip of a backbone carbonyl at G444 or Y445, displacing the bound ion from S2 and leading to pore collapse (Fig. 3 and Movie S4). In turn, collapse of the central pore increased the diameter of the peripheral cavity, allowing for faster water flow behind the selectivity filter into the intracellular vestibule of the channel pore (Movie S4).

Channel Block by Cs1 Is Slowed by Heavy Water.

We have further tested the proposed link between the aberrant mobility of water molecules behind the selectivity filter induced by the bound toxin to the observed asymmetric pore collapse in a series of simulations in which harmonic restraints were applied to the hydrogen bonds interconnecting these water molecules (SI Appendix, Table S3, trajectories 20 to 22, Fig. 5A and Movie S5). During these simulations, the pore conformation remained intact despite the bound toxin molecule (Fig. 5B and SI Appendix, Table S3). Analysis of the trajectory data has revealed that the applied restraints allowed the thermal agitation of water molecules within the peripheral pockets, but effectively prevented the infiltration of single water molecules through the aromatic cuff barrier. We have sought to experimentally emulate the simulated restraints on water mobility by exchanging D₂O for H₂O as the solvent during electrophysiological experiments. D₂O is highly compatible with electrophysiological measurements but has a lower diffusion coefficient compared with H₂O (by ∼20%; ref. 34) and forms deuterium bonds, which are more stable then hydrogen bonds (35). We have reasoned that these factors would result in an effective reduction of water molecule mobility at the peripheral pockets, reminiscent of the simulated restraints, thus allowing us to challenge experimentally the predicted reduction in toxin efficacy. To this end, we have employed the Shaker_KD M448K mutant, which exhibits accelerated slow inactivation while retaining a high affinity for toxins (SI Appendix, Fig. S5A and ref. 36). We first compared the rate of recovery from inactivation of this channel mutant in D₂O- and H₂O-based solutions (SI Appendix, Fig. S5B). We observed a small (18%), yet highly significant reduction in the fast component of recovery from slow inactivation upon transition from H₂O to D₂O, consistent with a slower rate of exchange of D₂O molecules buried behind the selectivity filter (33). We reasoned that while D₂O may affect channel gating via a nonspecific mechanism, resulting from its increased viscosity (37) or its effect on gating transitions (SI Appendix, Fig. S5E, see also ref. 38), since recovery from slow inactivation takes place at resting membrane potential, at which minimal gating transitions occur, it is unlikely to be altered by these nonspecific effects.

Fig. 5.

Channel block by Cs1 is slowed by heavy water. (A) Water occupancy in the peripheral pockets at the beginning of the restrained MD simulations (SI Appendix, Table S3, trajectories 20 to 22; Movie S5). The hydrogen bonds that interconnect these molecules (dotted lines) were restricted to their initial lengths by 5 kcal/mole harmonic restraints during these simulations. (B) Cross-subunit distance between the Cα atoms of G444 residues from chains A and C (red) or B and D (black) during restrained MD simulations. (C) Experimental design allowing determination of channel block kinetics with high temporal resolution. The voltage protocol is presented at the top; current traces illustrating a typical response are presented at the bottom. A control pulse (P₀) is applied to oocytes expressing Shaker_KD M448K, followed by application of a toxin dose inducing 80% to 90% block, and a first test pulse to +40 mV (P1). During P1, channels enter a slow-inactivated nonconductive state, and partial dissociation of the bound toxin takes place (SI Appendix, Fig. S5 C and D). A subsequent recovery period of variable duration (Δt) at holding membrane potential (−90 mV) allow channels to become available for reopening (τ = 0.2 s, SI Appendix, Fig. S6B), but also promote rebinding and block by the toxin (τ ∼ 2 s). These 2 opposing processes affect the current amplitude measured at P2, which exhibit a bell-shape kinetics (dashed line), with a decaying phase that reports the rate of block onset (τ₂, SI Appendix, Materials and Methods, Eq. 4). (D and E) Channel reblock kinetics measured in the presence of 20 nM ShK (Right) or 50 nM Cs1 (Left). I₁,I₂ are the current amplitudes recorded during P₁ and P₂, respectively. Data were collected in either H₂O-based (blue) or D₂O-based (red) solutions. Solid lines are best fits to Eq. 4; the derived time constants are summarized in SI Appendix, Table S4. The Insets display τ₂ values obtained from individual cells in H₂O (blue) or D₂O (red); solid lines connect data points derived from the same cell. For Cs1, τ₂ values were significantly higher in D₂O (Wilcoxon signed rank test, P = 0.03).

While Cs1 binding to unmodified Shaker_KD was voltage-insensitive (Fig. 2 C and D), we could partially reverse toxin block of the M448K mutant, using very long (>10 s) depolarizations (SI Appendix, Fig. S5C). This voltage-induced reversal of channel block was distinct from classical trans-enhanced dissociation, as it took place in the absence of an outward K⁺ current (SI Appendix, Fig. S5C and ref. 36). The toxin effect was restored upon subsequent incubation at resting membrane potential, at a rate that was toxin concentration dependent, giving rise to a bell-shaped curve, with a rising phase that follows recovery from slow inactivation and a falling phase that follows re-establishment of toxin block (Fig. 5C and SI Appendix, Fig. S5D). The rate constants associated with these 2 opposing processes typically differed by an order of magnitude, allowing for a straightforward isolation of Cs1 block onset rate with high temporal resolution. Cs1 block resettling curves revealed a clear decrease in rate upon transition from H₂O to D₂O (Fig. 5D), consistent with our prediction based on the restrained-MD simulations. This effect was specific; similar experiments conducted with the classic pore-blocker ShK did not reveal any effect of D₂O on block onset (Fig. 5E).

A Silent Binding Mode of Cs1 to a Noninactivating Shaker_KD Mutant.

The described mechanism of Cs1 action is highly reminiscent of the inherent slow inactivation, as both processes involve the constriction of the channel pore in response to alternating hydration patterns at the peripheral cavities. A mutation at the peripheral cavity upper barrier, T449Y, previously shown to eliminate slow inactivation (39), also abolished block by Cs1 (Fig. 6A). Yet, we did not assign this residue to the Cs1 binding site, as both mutagenesis and modeling data did not reveal any contribution of T449 for toxin binding. The finding that the T449Y mutation has not affected the binding of ShK (Fig. 6B), which makes close contact with the pore region, suggested that the substitution has not induced major rearrangements within the channel protein. To gain structural insight into the loss of function of Cs1 on Shaker_KD T449Y, we introduced the mutation into the docked toxin model and subjected the resulting complex to MD simulations. This complex retained all high-impact toxin-channel contacts and exhibited novel favorable interactions between the toxin and the substituted tyrosine (Fig. 6C). Simulation therefore predicts a tight binding of Cs1 to the Shaker_KD T449Y mutant, despite its apparent lack of activity. We tested this prediction by a series of functional binding competition assays in which the ability of Cs1 to inhibit the binding of ShK to Shaker_KD derivatives expressed in oocytes was examined (Fig. 6D). Indeed, we found that the rate of ShK association with Shaker_KD T449Y was reduced on preapplication of Cs1 (Fig. 6D and SI Appendix, Fig. S6 A and B). This effect was concentration dependent (Fig. 6D vs. SI Appendix, Fig. S6A) and specific for this channel mutant; we have not observed competition between the 2 toxins on a channel mutant with low affinity for Cs1 (F425A; Fig. 6 D, Middle) or on preapplication of a toxin mutant with low affinity for the channel (R49D; Fig. 6 D, Right). How could the apparent high-affinity binding of Cs1 to Shaker_KD T449Y be reconciled with its apparent lack of activity? MD simulations of the Cs1-bound T449Y channel mutant revealed high stability of the channel pore (SI Appendix, Fig. S6C). Analysis of these trajectories offered 2 nonredundant mechanisms (SI Appendix, Fig. S6 C–E) that rationalize the pore stability in the presence of bound toxin. The first is the stabilization of the aromatic cuff barrier by the newly introduced tyrosine rings. In subunits A and D of the channel, the Y449 rings rotated downward and formed multiple contacts with residues of the aromatic cuff barrier. This has stabilized the barrier and allowed it to resist the highly disordered water molecules allowed into the peripheral cavities by the bound toxin (SI Appendix, Fig. S6C and Movie S6). A second mechanism involved a structural rearrangement within the channel–toxin complex, in which the critical interactions between the C-terminal residues of the toxin and the D-W gates at chains C and D were replaced by a novel set of interactions with the newly introduced tyrosine rings (SI Appendix, Fig. S6 C–E and Movie S7). This rearrangement has diminished toxin effect on D-W gate dynamics and restored normal water permeation into the peripheral cavities. In summary, the silent binding of Cs1 to Shaker_KD T449Y strongly support our supposition that the toxin does not directly plug the ion conduction pathway, and simulations of the channel mutant with a bound toxin reveal specific modifications focused at the proposed action sites of the toxin.

Fig. 6.

A silent binding mode of Cs1 to a noninactivating Shaker mutant. (A and B) Dose–response curves of Cs1 (A) or ShK (B) determined on Shaker_KD derivatives expressed in oocytes. The EC₅₀ values obtained are 0.95 ± 0.03 nM (unmodified), 1.92 ± 0.64 (T449A), and >1 μM (T449Y) for Cs1 (A), and 0.29 ± 0.11 nM (unmodified), 0.68 ± 0.28 nM (F425A), 0.16 ± 0.06 (T449Y) for ShK (B). (C) Snapshot of the Cs1–Shaker_KD T449Y complex taken after 200 ns unconstrained MD simulation. Interacting residues are rendered as sticks, and channel pore-helices and the toxin are rendered as transparent ribbons. This structure retains all of the high-impact toxin-channel interactions identified by double-mutant cycle analysis (Fig. 1B). Note the newly introduced sets of cation–$\pi$ and $\pi$–$\pi$ interactions between the tyrosine substituted at position 449 and the C-terminal residues of the toxin. (D) Kinetics of Shaker_KD block by ShK alone or in the presence of Cs1. Peak K⁺ currents were recorded from oocytes expressing Shaker_KD T449Y (Left and Right) or F425A (Middle) for 60 s, and then 5 nM ShK was applied to the bath and the resulting current decay was recorded (red traces). In a coupled set of experiments conducted on oocytes from the same batch (blue traces), the introduction of ShK was preceded by the application of 50 nM Cs1 (Left and Middle) or 50 nM of the weakly active (EC₅₀ = 66.4 ± 5.4 nM) Cs1 R49D mutant (Right). Bars at the bottom indicate the timing of toxin application (BL, baseline). Shaded area behind the curves represents SD. Decay time constants calculated with (blue) and without (red) preapplication of Cs1 are given at the Insets. Statistical significance was determined using a Wilcoxon ranked-sum test (WT/T449Y; P = 0.01).

Discussion

Animal toxins acting on voltage gated ion channels were forged by evolution to rapidly alter their targets, endowing venomous organisms with powerful tools for predation and defense. These toxins have traditionally been categorized into 2 classes: gating modifier toxins, which alter ion conduction by interfering with the voltage-dependent motions of the channel, and pore blocker toxins that bind the channel pore and physically occlude the ion permeation pathway. Here, we present a mechanism used by animal toxins to block K⁺ channels, which defies the traditional classification and exposes a pharmacological target present in most ion channels.

Pore Modulation: A Block Mechanism of K⁺ Channels.

Classical pore blockers isolated from venomous organisms have played pivotal roles since the early days of ion channel research, facilitating the initial purification of novel channel proteins and providing the first insights into their subunit arrangement and the overall topology of the channel pore (33, 40, 41). Canonic K⁺ pore blockers display high phylogenetic diversity and variable backbone folds, yet they all share a strictly conserved lysine residue that physically plugs the channel pore (42). This mode of action was initially deduced from classical biophysical experiments (14) and later gained strong support upon the elucidation of a solid-state NMR (43) and crystal (17) structures of channel–toxin complexes. Alongside the canonic K⁺ blockers exists a diverse group of animal toxins that binds at the turret regions of K⁺ channels and blocks the ion conductance without physically occluding the pore (18–22). The precise molecular mechanisms by which toxins of this family block their targets have remained thus far elusive. Cs1, described here, clearly belongs to this second group of K⁺ blockers, and its mode of action, which we term pore-modulation, sheds light on the block mechanisms exploited by these toxins.

MD simulations of Shaker_KD in the presence of bound Cs1 revealed that in lack of tight binding between the toxin and the channel pore, water molecules and hydrated ions can access the extracellular vestibule (Fig. 2A). The demonstration that the noninactivating Shaker_KD T449Y mutant exhibits normal ion conduction while bound to Cs1 (Fig. 6) further negates a “pore-lid” mechanism for this toxin. Instead, we suggest a mechanism in which the contacts between Cs1 and the channel turrets serve primarily to coordinate its critical interactions with the D-W gates. These gates consist of a network of hydrogen bonds that govern water permeation into the water-filled peripheral cavities that surround the central ion pore. Toxin interference with the D-W gates creates imbalanced water traffic at the peripheral cavities and triggers pore collapse. These 3 principal properties of Cs1–turret binding, required for intact slow inactivation and interaction with the D-W gates, are shared by multiple K⁺ blocker families, for which a clear block mechanism has not been described: First, the cone snail toxins of the κM family that exhibit many pharmacological similarities to Cs1, despite their radically different backbone fold. κM-conotoxin RIIIK of this group binds the turret region of the Shaker channel, does not block the noninactivating T449Y mutant (44), and exhibit a ring of positively charged residues directed toward the channel pore (22). Second, the HERG blockers of the γ-KTX family. These scorpion toxins form multiple interactions with the extensive turrets of the HERG channel, yet their block is strongly affected by substitutions at the upper barrier that inhibit channel inactivation (19–21). The recent structure of one such channel mutant, HERG:S631A, clearly demonstrate that the substitution prevents a tilt of the aromatic ring of the signature motif and stabilize the canonic pore conformation (45). Our analysis argues strongly that these toxins, alongside other turret-binding toxins, block the channel using the water-mediated pore-modulation mechanism described here. Combined with recent reports of small molecules that bind at the peripheral cavities and modulate channel conductance (45, 46), pore-modulating toxins highlight these channel regions as an attractive pharmacological target and provides necessary cues for the rational design of compounds directed at these sites.

Pore Modulation and Slow Inactivation.

MD simulations of Cs1-bound Shaker_KD reveal a sequence of events that culminate in an asymmetric collapse of the channel pore, triggered by the bound toxin. Several recent studies conducted with unmodified and chemically modified channels suggest that the molecular events captured by our simulations faithfully describe channel dynamics. First, chemical modifications at the SF of KcsA that enhance slow inactivation were shown to trigger asymmetric collapse of the channel pore by altering the turnover of water molecules bound behind the SF. Basins at the free energy landscape of unmodified KcsA, corresponding to the asymmetrically constricted pore conformation, were detected (47). Second, a chemical modification of Kv1.2:W434 was shown to enhance slow inactivation by increasing water traffic through the D-W gate (48). A key event that reoccurred in all Cs1-bound simulations was the collapse of the hydrophobic cuff barrier that enabled water exchange between the peripheral pore and the central vestibule of the channel and between pockets of neighboring subunits (SI Appendix, Fig. S4 F and H and Movies S3 and S4). Flips of the pore backbone carbonyls then diverted this water stream into the main pore, leading to ion loss and rapid collapse of the SF. Flipped backbone carbonyls were captured in the crystal structures of inactivated ion channels (8). The recent Cryo-EM structures of the HERG channel and its noninactivating S631A mutant further establish a direct link between a compromised cuff-barrier and the inactivated state of the channel (45). Water conduction behind the collapsed SF of KcsA was suggested to explain the high water permeation of its closed state (49, 50). Our simulations reveal an opening of a hydrophilic path connecting the peripheral cavity with the intracellular vestibule that involves 2 conserved threonine sidechains (SI Appendix, Fig. S4F). Alanine substitutions at the second of these Thr residues were recently shown to impair slow inactivation in Kv1.2, Kv1.5, and KcsA and to alter the occupancy of water molecules buried behind the SF of crystalized KcsA mutants (51). Combined, these studies portray a molecular machinery composed of water paths and gates behind the K⁺ channel pore that set the rate and timing for slow inactivation. Pore modulating toxins efficiently exploit this machinery to impose ectopic channel gating.

Methods

Detailed descriptions of protein expression and purification, crystallization, and structural determination, electrophysiological data acquisition and analysis and molecular dynamics simulations are provided in SI Appendix, Supplementary Information Methods.

Data and Materials Availability.

Docked models of Cs1 and ShK alongside membrane and solvent are provided in PDB format as supplementary text files.