Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states.

In silico and in vitro studies have made progress in understanding protein-protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

Solution structure of extracellular loop of human ß4 subunit of BK channel and its biological implication on ChTX sensitivity.

Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming α subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BKα coexpression with auxiliary ß (ß1-ß4) subunits, as well as newly defined γ subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched ß4 subunit (hß4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (hß4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of ß4 subunit's extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by ß4 are still unclear. To address them, here, we first identified four disulfide bonds in hß4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of ß4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BKα/ß4 and thermodynamic double-mutant cycle analysis, we proposed that BKα subunit might interact with ß4 subunit through the conserved residue Glu264(BKα) coupling with residue Asn123(ß4).

Role of BKCa in Stretch-Induced Relaxation of Colonic Smooth Muscle.

Stretch-induced relaxation has not been clearly identified in gastrointestinal tract. The present study is to explore the role of large conductance calcium-activated potassium channels (BKCa) in stretch-induced relaxation of colon. The expression and currents of BKCa were detected and the basal muscle tone and contraction amplitude of colonic smooth muscle strips were measured. The expression of BKCa in colon is higher than other GI segments (P < 0.05). The density of BKCa currents was very high in colonic smooth muscle cells (SMCs). BKCa in rat colonic SMCs were sensitive to stretch. The relaxation response of colonic SM strips to stretch was attenuated by charybdotoxin (ChTX), a nonspecific BKCa blocker (P < 0.05). After blocking enteric nervous activities by tetrodotoxin (TTX), the stretch-induced relaxation did not change (P > 0.05). Still, ChTX and iberiotoxin (IbTX, a specific BKCa blocker) attenuated the relaxation of the colonic muscle strips enduring stretch (P < 0.05). These results suggest stretch-activation of BKCa in SMCs was involved in the stretch-induced relaxation of colon. Our study highlights the role of mechanosensitive ion channels in SMCs in colon motility regulation and their physiological and pathophysiological significance is worth further study.

Solid phase synthesis, NMR structure determination of α-KTx3.8, its in silico docking to Kv1.x potassium channels, and electrophysiological analysis provide insights into toxin-channel selectivity.

Animal venoms, such as those from scorpions, are a potent source for new pharmacological substances. In this study we have determined the structure of the α-KTx3.8 (named as Bs6) scorpion toxin by multidimensional (1)H homonuclear NMR spectroscopy and investigated its function by molecular dynamics (MD) simulations and electrophysiological measurements. Bs6 is a potent inhibitor of the Kv1.3 channel which plays an important role during the activation and proliferation of memory T-cells (TEM), which play an important role in autoimmune diseases. Therefore, it could be an interesting target for treatment of autoimmune diseases. In this study, Bs6 was synthesised by solid phase synthesis and its three-dimensional (3D) structure has been determined. To gain a deeper insight into the interaction of Bs6 with different potassium channels like hKv1.1 and hKv1.3, the protein-protein complex was modelled based on known toxin-channel structures and tested for stability in MD simulations using GROMACS. The toxin-channel interaction was further analysed by electrophysiological measurements of different potassium channels like hKv1.3 and hKv7.1. As potassium channel inhibitors could play an important role to overcome autoimmune diseases like multiple sclerosis and type-1 diabetes mellitus, our data contributes to the understanding of the molecular mechanism of action and will ultimately help to develop new potent inhibitors in future.

Molecular dynamics simulations of scorpion toxin recognition by the Ca(2+)-activated potassium channel KCa3.1.

The Ca(2+)-activated channel of intermediate-conductance (KCa3.1) is a target for antisickling and immunosuppressant agents. Many small peptides isolated from animal venoms inhibit KCa3.1 with nanomolar affinities and are promising drug scaffolds. Although the inhibitory effect of peptide toxins on KCa3.1 has been examined extensively, the structural basis of toxin-channel recognition has not been understood in detail. Here, the binding modes of two selected scorpion toxins, charybdotoxin (ChTx) and OSK1, to human KCa3.1 are examined in atomic detail using molecular dynamics (MD) simulations. Employing a homology model of KCa3.1, we first determine conduction properties of the channel using Brownian dynamics and ascertain that the simulated results are in accord with experiment. The model structures of ChTx-KCa3.1 and OSK1-KCa3.1 complexes are then constructed using MD simulations biased with distance restraints. The ChTx-KCa3.1 complex predicted from biased MD is consistent with the crystal structure of ChTx bound to a voltage-gated K(+) channel. The dissociation constants (Kd) for the binding of both ChTx and OSK1 to KCa3.1 determined experimentally are reproduced within fivefold using potential of mean force calculations. Making use of the knowledge we gained by studying the ChTx-KCa3.1 complex, we attempt to enhance the binding affinity of the toxin by carrying out a theoretical mutagenesis. A mutant toxin, in which the positions of two amino acid residues are interchanged, exhibits a 35-fold lower Kd value for KCa3.1 than that of the wild-type. This study provides insight into the key molecular determinants for the high-affinity binding of peptide toxins to KCa3.1, and demonstrates the power of computational methods in the design of novel toxins.

Two conserved arginine residues from the SK3 potassium channel outer vestibule control selectivity of recognition by scorpion toxins.

Potassium channel functions are often deciphered by using selective and potent scorpion toxins. Among these toxins, only a limited subset is capable of selectively blocking small conductance Ca(2+)-activated K(+) (SK) channels. The structural bases of this selective SK channel recognition remain unclear. In this work, we demonstrate the key role of the electric charges of two conserved arginine residues (Arg-485 and Arg-489) from the SK3 channel outer vestibule in the selective recognition by the SK3-blocking BmP05 toxin. Indeed, individually substituting these residues with histidyl or lysyl (maintaining the positive electric charge partially or fully), although decreasing BmP05 affinity, still preserved the toxin sensitivity profile of the SK3 channel (as evidenced by the lack of recognition by many other types of potassium channel-sensitive charybdotoxin). In contrast, when Arg-485 or Arg-489 of the SK3 channel was mutated to an acidic (Glu) or alcoholic (Ser) amino acid residue, the channel lost its sensitivity to BmP05 and became susceptible to the "new" blocking activity by charybdotoxin. In addition to these SK3 channel basic residues important for sensitivity, two acidic residues, Asp-492 and Asp-518, also located in the SK3 channel outer vestibule, were identified as being critical for toxin affinity. Furthermore, molecular modeling data indicate the existence of a compact SK3 channel turret conformation (like a peptide screener), where the basic rings of Arg-485 and Arg-489 are stabilized by strong ionic interactions with Asp-492 and Asp-518. In conclusion, the unique properties of Arg-485 and Arg-489 (spatial orientations and molecular interactions) in the SK3 channel account for its toxin sensitivity profile.

Charybdotoxin and margatoxin acting on the human voltage-gated potassium channel hKv1.3 and its H399N mutant: an experimental and computational comparison.

The effect of the pore-blocking peptides charybdotoxin and margatoxin, both scorpion toxins, on currents through human voltage-gated hK(v)1.3 wild-type and hK(v)1.3_H399N mutant potassium channels was characterized by the whole-cell patch clamp technique. In the mutant channels, both toxins hardly blocked current through the channels, although they did prevent C-type inactivation by slowing down the current decay during depolarization. Molecular dynamics simulations suggested that the fast current decay in the mutant channel was a consequence of amino acid reorientations behind the selectivity filter and indicated that the rigidity-flexibility in that region played a key role in its interactions with scorpion toxins. A channel with a slightly more flexible selectivity filter region exhibits distinct interactions with scorpion toxins. Our studies suggest that the toxin-channel interactions might partially restore rigidity in the selectivity filter and thereby prevent the structural rearrangements associated with C-type inactivation.

Rigid body Brownian dynamics as a tool for studying ion channel blockers.

Using a novel rigid body Brownian dynamics algorithm, we investigate how a spherically asymmetrical polyamine molecule, a branched analogue of spermine, interacts with the external vestibule of the voltage-gated potassium channel, Kv1.2. Simulations reveal that the blocker, with a charge of +4e, inserts one of its charged amine groups into the selectivity filter, while another forms a salt bridge with an aspartate residue located just outside the entrance of the pore. This binding mode mimics features of the binding of polypeptides such as the scorpion venom charybdotoxin to the channel. The potential of mean force constructed with Brownian dynamics is a reasonable match to that obtained from molecular dynamics simulations, with dissociation constants of 4.7 and 22 µM, respectively. The current-voltage relationships obtained with and without a blocker in the external reservoir show that the inward current is severely attenuated by the presence of the blocker, whereas the outward current is only moderately reduced. The computational molecular modeling technique we introduce here can provide detailed insights into ligand-channel interactions and can be used for rapidly screening potential blocker molecules.

Charybdotoxin unbinding from the mKv1.3 potassium channel: a combined computational and experimental study.

Charybdotoxin, belonging to the group of so-called scorpion toxins, is a short peptide able to block many voltage-gated potassium channels, such as mKv1.3, with high affinity. We use a reliable homology model based on the high-resolution crystal structure of the 94% sequence identical homologue Kv1.2 for charybdotoxin docking followed by molecular dynamics simulations to investigate the mechanism and energetics of unbinding, tracing the behavior of the channel protein and charybdotoxin during umbrella-sampling simulations as charybdotoxin is moved away from the binding site. The potential of mean force is constructed from the umbrella sampling simulations and combined with K(d) and free energy values gained experimentally using the patch-clamp technique to study the free energy of binding at different ion concentrations and the mechanism of the charybdotoxin-mKv1.3 binding process. A possible charybdotoxin binding mechanism is deduced that includes an initial hydrophobic contact followed by stepwise electrostatic interactions and finally optimization of hydrogen bonds and salt bridges.

Accurate determination of the binding free energy for KcsA-charybdotoxin complex from the potential of mean force calculations with restraints.

Free energy calculations for protein-ligand dissociation have been tested and validated for small ligands (50 atoms or less), but there has been a paucity of studies for larger, peptide-size ligands due to computational limitations. Previously we have studied the energetics of dissociation in a potassium channel-charybdotoxin complex by using umbrella sampling molecular-dynamics simulations, and established the need for carefully chosen coordinates and restraints to maintain the physiological ligand conformation. Here we address the ligand integrity problem further by constructing additional potential of mean forces for dissociation of charybdotoxin using restraints. We show that the large discrepancies in binding free energy arising from simulation artifacts can be avoided by using appropriate restraints on the ligand, which enables determination of the binding free energy within the chemical accuracy. We make several suggestions for optimal choices of harmonic potential parameters and restraints to be used in binding studies of large ligands.