A set of homology models of pore loop domain of six eukaryotic voltage-gated potassium channels Kv1.1-Kv1.6

Homology models of the pore loop domain of six eukaryotic potassium channels, Kv1.1-Kv1.6, were generated based on the crystallographic structure of KcsA. The results of amino acid sequence alignment indicate that these Kv channels are composed of two structurally and functionally independent domains: the N-terminal 'voltage sensor' domain and the C-terminal 'pore loop' domain. The homology models reveal that the pore loop domains of these Kv channels exhibit similar folds to those of KcsA. The structural features and specific packing of aromatic residues around the selectivity filter of these Kv channels are nearly identical to those of KcsA, whereas most of the structural variations occur in the turret as well as in the inner and outer helices. The distribution of polar and nonpolar side chains on the surfaces of the KcsA and Kv channels reveals that they exhibit a segregation of side chains common to most integral membrane proteins. As the hydrogen bond between Glu71 and Asp80 in KcsA plays an important role in stabilizing the channel, the substituted Val residue in the Kv family corresponding to Glu71 of KcsA stabilizes the channel by making hydrophobic contact with Tyr residue from the signature sequence of the selectivity filter. The homology models of these Kv channels provide particularly attractive subjects for further structure-based studies.     

Molecular Docking of the Scorpion Toxin Tc1 to the Structural Model of the Voltage-gated Potassium Channel Kv1.1 from Human Homo sapiens

Abstract

In this study, structural model of the pore loop region of the voltage-gated potassium channel Kv1.1 from human Homo sapiens was constructed based on the crystallographic structure of KcsA by structural homology. The pore loop region of Kv1.1 exhibits similar folds as that of KcsA. The structural feature of the selectivity filter of Kv1.1 is nearly identical to that of KcsA, whereas most of the structural variations occur in the turret as well as in the inner and outer helices. Molecular docking experiments of the scorpion toxin Tc1 from Tityus cambridgei to the outer vestibule of KcsA as well as Kv1.1 were subsequently performed with various initial Tc1 orientations. Tc1 was found to form the most stable complexes with these two K⁺ channels when the side chain of Lys14 occupies the pore of the selectivity filter through electrostatic interaction. Tc1 binds preferentially towards Kv1.1 than KcsA due to stronger hydrophobic and electrostatic interactions formed between the toxin and the selectivity filter and outer vestibule of Kv1.1. Furthermore, surface complementarity of the outer vestibules of the channels to the Tc1 spatial conformations also plays an important role in stabilizing both the Tc1/KcsA and Tc1/Kv1.1 complexes.     

Molecular docking of the scorpion toxin Tc1 to the structural model of the voltage-gated potassium channel Kv1.1 from human Homo sapiens

In this study, structural model of the pore loop region of the voltage-gated potassium channel Kv1.1 from human Homo sapiens was constructed based on the crystallographic structure of KcsA by structural homology. The pore loop region of Kv1.1 exhibits similar folds as that of KcsA. The structural feature of the selectivity filter of Kv1.1 is nearly identical to that of KcsA, whereas most of the structural variations occur in the turret as well as in the inner and outer helices. Molecular docking experiments of the scorpion toxin Tc1 from Tityus cambridgei to the outer vestibule of KcsA as well as Kv1.1 were subsequently performed with various initial Tc1 orientations. Tc1 was found to form the most stable complexes with these two K+ channels when the side chain of Lys14 occupies the pore of the selectivity filter through electrostatic interaction. Tc1 binds preferentially towards Kv1.1 than KcsA due to stronger hydrophobic and electrostatic interactions formed between the toxin and the selectivity filter and outer vestibule of Kv1.1. Furthermore, surface complementarity of the outer vestibules of the channels to the Tc1 spatial conformations also plays an important role in stabilizing both the Tc1/KcsA and Tc1/Kv1.1 complexes.     

Diverse gating in K channels: Differential role of the pore-helix glutamate in stabilizing the channel pore

The selectivity filter and adjacent regions in the bacterial KcsA and inwardly rectifying K⁺ (Kir) channels reveal significant conformational changes that cause the channel pore to transition from an activated to inactive state (C-type inactivation) once the channel is open. The meshwork of residues stabilizing the pore of KcsA involves Glu71–Asp80 carboxyl–carboxylate interaction ‘behind’ the selectivity filter. Interestingly, the Kir channels do not have this exact interaction, but instead have a Glu–Arg salt bridge where the Glu is in the same position but the Arg is one position N-terminal compared to the Asp in KcsA. Also, the Kir channels lack the Trp that hydrogen bonds to Asp80 in KcsA. Here, the sequence and structural information are combined to understand the dissimilarity in the role of the pore-helix Glu in stabilizing the pore structure in KcsA and Kir channels. This review illustrates that although Glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other; thereby suggesting a unique phenomenon of diverse gating patterns in K⁺ channels.     

The investigation of interactions of kappa-Hefutoxin1 with the voltage-gated potassium channels: a computational simulation

Kappa-Hefutoxin1 is a K(+) channel-blocking toxin from the scorpion Heterometrus fluvipes. It is a 22-residue protein that adapts a novel fold of two parallel helices linked by two disulfide bridges without beta-sheets. Recognition of interactions of kappa-Hefutoxin1 with the voltage-gated potassium channels, Kv1.1, Kv1.2, and Kv1.3, was studied by 3D-Dock software package. All structures of kappa-Hefutoxin1 were considered during the simulations, which indicated that even small changes in the structure of kappa-Hefutoxin1 considerably affected both the recognition and the binding between kappa-Hefutoxin1 and the Kv1 channels. kappa-Hefutoxin1 is located around the extracellular part of the Kv1 channels, making contacts with its helices. Lys 19, Tyr 5, Arg 6, Trp 9, or Arg 10 in the toxin and residues Asp 402, His 404, Thr 407,Gly 401, and Asp 386 in each subunit of the Kv potassium channel are the key residues for the toxin-channel recognition. Moreover, the simulation result demonstrates that the hydrophobic interactions are important in interaction of negatively charged toxins with potassium channels. The results of our docking/molecular dynamics simulations indicate that our 3D model structure of the kappa-Hefutoxin1-complex is both reasonable and acceptable and could be helpful for smarter drug design and the blocking agents of Kv1 channels.     

Brownian dynamics simulations of the recognition of the scorpion toxin maurotoxin with the voltage-gated potassium ion channels

The recognition of the scorpion toxin maurotoxin (MTX) by the voltage-gated potassium (Kv1) channels, Kv1.1, Kv1.2, and Kv1.3, has been studied by means of Brownian dynamics (BD) simulations. All of the 35 available structures of MTX in the Protein Data Bank (http://www.rcsb.org/pdb) determined by nuclear magnetic resonance were considered during the simulations, which indicated that the conformation of MTX significantly affected both the recognition and the binding between MTX and the Kv1 channels. Comparing the top five highest-frequency structures of MTX binding to the Kv1 channels, we found that the Kv1.2 channel, with the highest docking frequencies and the lowest electrostatic interaction energies, was the most favorable for MTX binding, whereas Kv1.1 was intermediate, and Kv1.3 was the least favorable one. Among the 35 structures of MTX, the 10th structure docked into the binding site of the Kv1.2 channel with the highest probability and the most favorable electrostatic interactions. From the MTX-Kv1.2 binding model, we identified the critical residues for the recognition of these two proteins through triplet contact analyses. MTX locates around the extracellular mouth of the Kv1 channels, making contacts with its beta-sheets. Lys23, a conserved amino acid in the scorpion toxins, protrudes into the pore of the Kv1.2 channel and forms two hydrogen bonds with the conserved residues Gly401(D) and Tyr400(C) and one hydrophobic contact with Gly401(C) of the Kv1.2 channel. The critical triplet contacts for recognition between MTX and the Kv1.2 channel are Lys23(MTX)-Asp402(C)(Kv1), Lys27(MTX)-Asp378(D)(Kv1), and Lys30(MTX)-Asp402(A)(Kv1). In addition, six hydrogen-bonding interactions are formed between residues Lys23, Lys27, Lys30, and Tyr32 of MTX and residues Gly401, Tyr400, Asp402, Asp378, and Thr406 of Kv1.2. Many of them are formed by side chains of residues of MTX and backbone atoms of the Kv1.2 channel. Five hydrophobic contacts exist between residues Pro20, Lys23, Lys30 and Tyr32 of MTX and residues Asp402, Val404, Gly401, and Arg377 of the Kv1.2 channel. The simulation results are in agreement with the previous molecular biology experiments and explain the binding phenomena between MTX and Kv1 channels at the molecular level. The consistency between the results of the BD simulations and the experimental data indicated that our three-dimensional model of the MTX-Kv1.2 channel complex is reasonable and can be used in additional biological studies, such as rational design of novel therapeutic agents blocking the voltage-gated channels and in mutagenesis studies in both the toxins and the Kv1 channels. In particular, both the BD simulations and the molecular mechanics refinements indicate that residue Asp378 of the Kv1.2 channel is critical for its recognition and binding functionality toward MTX. This phenomenon has not been appreciated in the previous mutagenesis experiments, indicating this might be a new clue for additional functional study of Kv1 channels.     

Effects of Kv1.2 intracellular regions on activation of Kv2.1 channels

Depolarizing voltage steps activate voltage-dependent K(+) (Kv) channels by moving the voltage sensor, which triggers a coupling reaction leading to the opening of the pore. We constructed chimeric channels in which intracellular regions of slowly activating Kv2.1 channels were replaced by respective regions of rapidly activating Kv1.2 channels. Substitution of either the N-terminus, S4-S5 linker, or C-terminus generated chimeric Kv2.1/1.2 channels with a paradoxically slow and approximately exponential activation time course consisting of a fast and a slow component. Using combined chimeras, each of these Kv1.2 regions further slowed activation at the voltage of 0 mV, irrespective of the nature of the other two regions, whereas at the voltage of 40 mV both slowing and accelerating effects were observed. These results suggest voltage-dependent interactions of the three intracellular regions. This observation was quantified by double-mutant cycle analysis. It is concluded that interactions between N-terminus, S4-S5 linker, and/or C-terminus modulate the activation time course of Kv2.1 channels and that part of these interactions is voltage dependent.     

KChAP/Kvbeta1.2 interactions and their effects on cardiac Kv channel expression

KChAP and voltage-dependent K+ (Kv) beta-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta-subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta1.2, and Kvbeta1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.     

K+ conduction in the selectivity filter of potassium channels is monitored by the charge distribution along their sequence

Potassium channels display a high conservation of sequence of the selectivity filter (SF), yet nature has designed a variety of channels that present a wide range of absolute rates of K(+) permeation. In KcsA, the structural archetype for K channels, under physiological concentrations, two K(+) ions reside in the SF in configurations 1,3 (up state) and 2,4 (down state) and ion conduction is believed to follow a throughput cycle involving a transition between these states. Using free-energy calculations of KcsA, Kv1.2, and mutant channels, we show that this transition is characterized by a channel-dependent energy barrier. This barrier is strongly influenced by the charges partitioned along the sequence of each channel. These results unveil therefore how, for similar structures of the SF, the rate of K(+) turnover may be fine-tuned within the family of potassium channels.     

Molecular basis of inhibitory peptide maurotoxin recognizing Kv1.2 channel explored by ZDOCK and molecular dynamic simulations

Inhibitory peptide-channel interactions have been utilized to characterize both channels and peptides; however, the fundamental basis for these interactions remains elusive. Here, combined computation methods were employed to study the specific binding of maurotoxin (MTX) peptide to Kv1.2 channel. In the first stage, numerous predicted complexes were generated by docking an ensemble of all 35 NMR conformations of MTX to Kv1.2 channel with ZDOCK program. Then the resulted complexes were clustered and classified into four main binding modes, based on experimental information and interaction energy analysis after the energy minimization and molecular dynamics (MD) simulations. By examining the stability of the plausible candidates through unrestrained MD simulations and calculation of the binding free energies, a final reasonable MTX-Kv1.2 complex was identified, with an overall high degree of correlation between the calculation and experiment on mutational effects. In the obtained complex structure model, MTX mainly used its beta-sheet domains to associate the channel mouth instead of the well-recognized functionally important S5P linkers of Kv1.2 channel. Structure analysis characterized that the most essential Tyr(32) residue of MTX was surrounded by a "pocket" formed by many nonpolar and polar residues of Kv1.2 channel, and revealed a pore-blocking Lys(23) and an important Lys(7) stabilized by strong electrostatic interactions with Asp(379) of Kv1.2. Furthermore, a stepwise structural arrangement for both ligand and receptor was found to accompany the tighter interaction of MTX into the target channel. The starting conformation of MTX, the side-chain conformation of the most important residue Tyr(32), and proper introduction of flexibility for candidate complexes were demonstrated to be considerably important factors for obtaining the final reasonable complex structure model. All these findings should not only be helpful for identifying more plausible K(+) channel-inhibitory peptide complex structures, but also provide intrinsically valuable structural biology information to interpret binding affinities, specificities, and diversity of K(+) channel-nature toxin interactions.     

Generating a high affinity scorpion toxin receptor in KcsA-Kv1.3 chimeric potassium channels

The crystal structure of the bacterial K(+) channel, KcsA (Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77), and subsequent mutagenesis have revealed a high structural conservation from bacteria to human (MacKinnon, R., Cohen, S. L., Kuo, A., Lee, A., and Chait, B. T. (1998) Science 280, 106-109). We have explored this conservation by swapping subregions of the M1-M2 linker of KcsA with those of the S5-S6 linker of the human Kv-channel Kv1.3. The chimeric K(+) channel constructs were expressed in Escherichia coli, and their multimeric state was analyzed after purification. We used two scorpion toxins, kaliotoxin and hongotoxin 1, which bind specifically to Kv1.3, to analyze the pharmacological properties of the KcsA-Kv1.3 chimeras. The results demonstrate that the high affinity scorpion toxin receptor of Kv1.3 could be transferred to KcsA. Our biochemical studies with purified KcsA-Kv1.3 chimeras provide direct chemical evidence that a tetrameric channel structure is necessary for forming a functional scorpion toxin receptor. We have obtained KcsA-Kv1.3 chimeras with kaliotoxin affinities (IC(50) values of approximately 4 pm) like native Kv1.3 channels. Furthermore, we show that a subregion of the S5-S6 linker may be an important determinant of the pharmacological profile of K(+) channels. Using available structural information on KcsA and kaliotoxin, we have developed a structural model for the complex between KcsA-Kv1.3 chimeras and kaliotoxin to aid future pharmacological studies of K(+) channels.     

K Conduction in the Selectivity Filter of Potassium Channels Is Monitored by the Charge Distribution along Their Sequence

Potassium channels display a high conservation of sequence of the selectivity filter (SF), yet nature has designed a variety of channels that present a wide range of absolute rates of K⁺ permeation. In KcsA, the structural archetype for K channels, under physiological concentrations, two K⁺ ions reside in the SF in configurations 1,3 (up state) and 2,4 (down state) and ion conduction is believed to follow a throughput cycle involving a transition between these states. Using free-energy calculations of KcsA, Kv1.2, and mutant channels, we show that this transition is characterized by a channel-dependent energy barrier. This barrier is strongly influenced by the charges partitioned along the sequence of each channel. These results unveil therefore how, for similar structures of the SF, the rate of K⁺ turnover may be fine-tuned within the family of potassium channels.     

Mixed modes in opening of KcsA potassium channel from a targeted molecular dynamics simulation

Potassium channels conduct K⁺ flow selectively across the membrane through a central pore. During a process called gating, the potassium channels undergo a conformational change that opens or closes the ion-conducting pore. The potassium channel KcsA has been structurally determined in its closed state. However, the dynamic mechanism of the gating transition of the KcsA channel is still being investigated. Here, a targeted molecular dynamics simulation up to 150 ns is performed to investigate the detailed opening process of the KcsA channel with an open Kv1.2 structure serving as the target. The channel arrived at a self-determined quasi-stable state within 60 ns. The rigid-body and hinge-bending modes are observed mixed together in the remaining 90 ns long quasi-stable state. The mixed-mode movement seems come from the competition between the helix rigidity and the biased-applied gating force.     

Identification of residues in dendrotoxin K responsible for its discrimination between neuronal K+ channels containing Kv1.1 and 1.2 alpha subunits.

Dendrotoxin (DTX) homologues are powerful blockers of K+ channels that contain certain subfamily Kv1 (1.1-1.6) alpha- and beta-subunits, in (alpha)4(beta)4 stoichiometry. DTXk inhibits potently Kv1.1-containing channels only, whereas alphaDTX is less discriminating, but exhibits highest affinity for Kv1.2. Herein, the nature of interactions of DTXk with native K+ channels composed of Kv1.1 and 1.2 (plus other) subunits were examined, using 15 site-directed mutants in which amino acids were altered in the 310-helix, beta-turn, alpha-helix and random-coil regions. The mutants' antagonism of high-affinity [125I]DTXk binding to Kv1. 1-possessing channels in rat brain membranes and blockade of the Kv1. 1 current expressed in oocytes were quantified. Also, the levels of inhibition of [125I]alphaDTX binding to brain membranes by the DTXk mutants were used to measure their high- and low-affinity interactions, respectively, with neuronal Kv1.2-containing channels that possess Kv1.1 as a major or minor constituent. Displacement of toxin binding to either of these subtypes was not altered by single substitution with alanine of three basic residues in the random-coil region, or R52 or R53 in the alpha-helix; accordingly, representative mutants (K17A, R53A) blocked the Kv1.1 current with the same potency as the natural toxin. In contrast, competition of the binding of the radiolabelled alphaDTX or DTXk was dramatically reduced by alanine substitution of K26 or W25 in the beta-turn whereas changing nearby residues caused negligible alterations. Consistently, W25A and K26A exhibited diminished functional blockade of the Kv1.1 homo-oligomer. The 310-helical N-terminal region of DTXk was found to be responsible for recognition of Kv1.1 channels because mutation of K3A led to approximately 1246-fold reduction in the inhibitory potency for [125I]DTXk binding and a large decrease in its ability to block the Kv1.1 current; the effect of this substitution on the affinity of DTXk for Kv1.2-possessing oligomers was much less dramatic (approximately 16-fold).     

Position-dependent attenuation by Kv1.6 of N-type inactivation of Kv1.4-containing channels

Assembly of distinct α subunits of Kv1 (voltage-gated K(+) channels) into tetramers underlies the diversity of their outward currents in neurons. Kv1.4-containing channels normally exhibit N-type rapid inactivation, mediated through an NIB (N-terminal inactivation ball); this can be over-ridden if associated with a Kv1.6 α subunit, via its NIP (N-type inactivation prevention) domain. Herein, NIP function was shown to require positioning of Kv1.6 adjacent to the Kv1.4 subunit. Using a recently devised gene concatenation, heterotetrameric Kv1 channels were expressed as single-chain proteins on the plasmalemma of HEK (human embryonic kidney)-293 cells, so their constituents could be arranged in different positions. Placing the Kv1.4 and 1.6 genes together, followed by two copies of Kv1.2, yielded a K(+) current devoid of fast inactivation. Mutation of critical glutamates within the NIP endowed rapid inactivation. Moreover, separating Kv1.4 and 1.6 with a copy of Kv1.2 gave a fast-inactivating K(+) current with steady-state inactivation shifted to more negative potentials and exhibiting slower recovery, correlating with similar inactivation kinetics seen for Kv1.4-(1.2)(3). Alternatively, separating Kv1.4 and 1.6 with two copies of Kv1.2 yielded slow-inactivating currents, because in this concatamer Kv1.4 and 1.6 should be together. These findings also confirm that the gene concatenation can generate K(+) channels with α subunits in pre-determined positions.     

Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca(2+)-activated over voltage-gated K(+) channels

We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca(2+)-activated K(+) channels, into a selective inhibitor. As a model system, we chose two channels in T-lymphocytes, the voltage-gated channel Kv1.3 and the Ca(2+)-activated channel IKCa1. Homology models of both channels were generated based on the crystal structure of the bacterial channel KcsA. Initial docking of charybdotoxin was undertaken with both models, and the accuracy of these docking configurations was tested by mutant cycle analyses, establishing that charybdotoxin has a similar docking configuration in the external vestibules of IKCa1 and Kv1.3. Comparison of the refined models revealed a unique cluster of negatively charged residues in the turret of Kv1.3, not present in IKCa1. To exploit this difference, three novel charybdotoxin analogs were designed by introducing negatively charged residues in place of charybdotoxin Lys(32), which lies in close proximity to this cluster. These analogs block IKCa1 with approximately 20-fold higher affinity than Kv1.3. The other charybdotoxin-sensitive Kv channels, Kv1.2 and Kv1. 6, contain the negative cluster and are predictably insensitive to the charybdotoxin position 32 analogs, whereas the maxi-K(Ca) channel, hSlo, lacking the cluster, is sensitive to the analogs. This provides strong evidence for topological similarity of the external vestibules of diverse K(+) channels and demonstrates the feasibility of using structure-based strategies to design selective inhibitors for mammalian K(+) channels. The availability of potent and selective inhibitors of IKCa1 will help to elucidate the role of this channel in T-lymphocytes during the immune response as well as in erythrocytes and colonic epithelia.     

Ion selectivity in potassium channels

Potassium channels are tetrameric membrane-spanning proteins that provide a selective pore for the conduction of K(+) across the cell membranes. One of the main physiological functions of potassium channels is efficient and very selective transport of K(+) ions through the membrane to the cell. Classical views of ion selectivity are summarized within a historical perspective, and contrasted with the molecular dynamics (MD) simulations free energy perturbation (FEP) performed on the basis of the crystallographic structure of the KcsA phospholipid membrane. The results show that the KcsA channel does not select for K(+) ions by providing a binding site of an appropriate (fixed) cavity size. Rather, selectivity for K(+) arises directly from the intrinsic local physical properties of the ligands coordinating the cation in the binding site, and is a robust feature of a pore symmetrically lined by backbone carbonyl groups. Further analysis reveals that it is the interplay between the attractive ion-ligand (favoring smaller cation) and repulsive ligand-ligand interactions (favoring larger cations) that is the basic element governing Na(+)/K(+) selectivity in flexible protein binding sites. Because the number and the type of ligands coordinating an ion directly modulate such local interactions, this provides a potent molecular mechanism to achieve and maintain a high selectivity in protein binding sites despite a significant conformational flexibility.     

Mutating a critical lysine in ShK toxin alters its binding configuration in the pore-vestibule region of the voltage-gated potassium channel,Kv1.3

The voltage-gated potassium channel in T lymphocytes, Kv1.3, an important target for immunosuppressants, is blocked by picomolar concentrations of the polypeptide ShK toxin and its analogue ShK-Dap22. ShK-Dap22 shows increased selectivity for Kv1.3, and our goal was to determine the molecular basis for this selectivity by probing the interactions of ShK and ShK-Dap22 with the pore and vestibule of Kv1.3. The free energies of interactions between toxin and channel residues were measured using mutant cycle analyses. These data, interpreted as approximate distance restraints, guided molecular dynamics simulations in which the toxins were docked with a model of Kv1.3 based on the crystal structure of the bacterial K(+)-channel KcsA. Despite the similar tertiary structures of the two ligands, the mutant cycle data imply that they make different contacts with Kv1.3, and they can be docked with the channel in configurations that are consistent with the mutant cycle data for each toxin but quite distinct from one another. ShK binds to Kv1.3 with Lys22 occupying the negatively charged pore of the channel, whereas the equivalent residue in ShK-Dap22 interacts with residues further out in the vestibule, producing a significant change in toxin orientation. The increased selectivity of ShK-Dap22 is achieved by strong interactions of Dap22 with His404 and Asp386 on Kv1.3, with only weak interactions between the channel pore and the toxin. Potent and specific blockade of Kv1.3 apparently occurs without insertion of a positively charged residue into the channel pore. Moreover, the finding that a single residue substitution alters the binding configuration emphasizes the need to obtain consistent data from multiple mutant cycle experiments in attempts to define protein interaction surfaces using these data.