New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1

The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.     

BeKm-1 is a HERG-specific toxin that shares the structure with ChTx but the mechanism of action with ErgTx1

Peptide toxins with disulfide-stabilized structures have been used as molecular calipers to probe the outer vestibule structure of K channels. We want to apply this approach to the human ether-a-go-go-related gene (HERG) channel, whose outer vestibule is unique in structure and function among voltage-gated K channels. Our focus here is BeKm-1, a HERG-specific peptide toxin that can suppress HERG in the low nM concentration range. Although BeKm-1 shares the three-dimensional scaffold with the well-studied charybdotoxin, the two use different mechanisms in suppressing currents through their target K channels. BeKm-1 binds near, but not inside, the HERG pore, and it is possible that BeKm-1-bound HERG channels can conduct currents although with markedly altered voltage-dependence and kinetics of gating. BeKm-1 and ErgTx1 differ in three-dimensional scaffold, but the two share mechanism of action and have overlapping binding sites on the HERG channel. For both, residues in the middle of the S5-P linker (the putative 583-597 helix) and residues at the pore entrance are critical for binding, although specific contact points vary between the two. Toxin foot printing using BeKm-1 and ErgTx1 will likely provide complementary information about the unique outer vestibule structure of the HERG channel.     

Interaction simulation of hERG K+ channel with its specific BeKm-1 peptide: insights into the selectivity of molecular recognition

Potassium channels show a huge variability in the affinity when recognizing enormous bioactive peptides, and the elucidation of their recognition mechanism remains a great challenge due to an undetermined peptide-channel complex structure. Here, we employed combined computation methods to study the specific binding of BeKm-1 peptide to the hERG potassium channel, which is an essential determinant of the long-QT syndrome. By the use of a segment-assembly homology modeling method, the closed-state hERG structure containing unusual longer S5P linker was successfully constructed. It has a "petunia" shape, while four "petals" of symmetrically distributed S5P segments always decentralize. Starting from the hERG and BeKm-1 structures, a considerably reasonable BeKm-1-hERG complex structure was then screened out and identified by protein-protein docking, molecular dynamics (MD) simulations, and calculation of relative binding free energies. The validity of this predicted complex was further assessed by computational alanine-scanning, with the results correlating reasonably well with experimental data. In the novel complex structure, four considerably flexible S5P linkers are far from the BeKm-1 peptide. The BeKm-1 mainly uses its helical region to associate the channel outer vestibule, except for the S5P linker region; however, structural analysis further implies this neutral pore region with wiggling S5P linker is highly beneficial to the binding of BeKm-1 with lower positive charges. The most critical Lys18 of BeKm-1 plugs its side chain into the channel selectivity filter, while the secondarily important Arg20 forms three hydrogen bonds with spatially neighboring residues in the hERG channel. Different from the classical peptide-K+ channel interaction mainly induced by electrostatic interaction, a synergetic effect of the electrostatic and van der Waals interactions was found to mediate the molecular recognition between BeKm-1 and the hERG channel. And this specific binding process is revealed to be a dynamic change of reduction of binding free energy and conformational rearrangement mainly in the interface of both BeKm-1 and the hERG channel. All these structural and energy features yield deep insights on the high selective binding mechanism of hERG-specific peptides, present a diversity of peptide-K+ channel interactions, and also provide important clues to further study structure-function relationships of the hERG channel.     

Probing the outer mouth structure of the HERG channel with peptide toxin footprinting and molecular modeling

Previous studies have shown that the unusually long S5-P linker lining human ether a-go-go related gene's (hERG's) outer vestibule is critical for its channel function: point mutations at high-impact positions here can interfere with the inactivation process and, in many cases, also reduce the pore's K+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three-dimensional structure. The first part of this article describes mutant cycle analysis used to identify residues on hERG's outer vestibule that interact with specific residues on the interaction surface of BeKm-1, a peptide toxin with known NMR structure and a high binding affinity to hERG. The second part describes molecular modeling of hERG's pore domain. The transmembrane region was modeled after the crystal structure of KvAP pore domain. The S5-P linker was docked to the transmembrane region based on data from previous NMR and mutagenesis experiments, as well as a set of modeling criteria. The models were further restrained by contact points between hERG's outer vestibule and the bound BeKm-1 toxin molecule deduced from the mutant cycle analysis. Based on these analyses, we propose a working model for the open conformation of the outer vestibule of the hERG channel, in which the S5-P linkers interact with the pore loops to influence ion flux through the pore.     

Structural and functional role of the extracellular s5-p linker in the HERG potassium channel

C-type inactivation in the HERG channel is unique among voltage-gated K channels in having extremely fast kinetics and strong voltage sensitivity. This suggests that HERG may have a unique outer mouth structure (where conformational changes underlie C-type inactivation), and/or a unique communication between the outer mouth and the voltage sensor. We use cysteine-scanning mutagenesis and thiol-modifying reagents to probe the structural and functional role of the S5-P (residues 571-613) and P-S6 (residues 631-638) linkers of HERG that line the outer vestibule of the channel. Disulfide formation involving introduced cysteine side chains or modification of side chain properties at "high-impact" positions produces a common mutant phenotype: disruption of C-type inactivation, reduction of K+ selectivity, and hyperpolarizing shift in the voltage-dependence of activation. In particular, we identify 15 consecutive positions in the middle of the S5-P linker (583-597) where side chain modification has marked impact on channel function. Analysis of the degrees of mutation-induced perturbation in channel function along 583-597 reveals an alpha-helical periodicity. Furthermore, the effects of MTS modification suggest that the NH2-terminal of this segment (position 584) may be very close to the pore entrance. We propose a structural model for the outer vestibule of the HERG channel, in which the 583-597 segment forms an alpha-helix. With the NH2 terminus of this helix sitting at the edge of the pore entrance, the length of the helix (approximately 20 A) allows its other end to reach and interact with the voltage-sensing domain. Therefore, the "583-597 helix" in the S5-P linker of the HERG channel serves as a bridge of communication between the outer mouth and the voltage sensor, that may make important contribution to the unique C-type inactivation phenotype.     

The S631A mutation causes a mechanistic switch in the block of hERG channels by CnErg1

We have studied the interaction of CnErg1, a member of the gamma-KTX subfamily of scorpion toxins with the inactivation-deficient S631A hERG channel. In the background of this mutation, we observed a mechanistic switch from turret block, characteristic of the action of gamma-KTXs on Kv11-type channels, to pore plugging, characteristic of alpha-KTX block of Kv1-type channels. We suggest this reflects destabilization of the outer pore (turret region) of hERG allowing access of the toxin molecule to directly plug the conduction pathway.     

An ERG channel inhibitor from the scorpion Buthus eupeus

The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.     

Potential role of the membrane in hERG channel functioning and drug-induced long QT syndrome

The human ether-à-go-go related gene (hERG) potassium channels are located in the myocardium cell membrane where they ensure normal cardiac activity. The binding of drugs to this channel, a side effect known as drug-induced (acquired) long QT syndrome (ALQTS), can lead to arrhythmia or sudden cardiac death. The hERG channel is a unique member of the family of voltage-gated K⁺ channels because of the long extracellular loop connecting its transmembrane S5 helix to the pore helix in the pore domain. Considering the proximal position of the S5-P linker to the membrane surface, we have investigated the interaction of its central segment I⁵⁸³-Y⁵⁹⁷ with bicelles. Liquid and solid-state NMR experiments as well as circular dichroism results show a strong affinity of the I⁵⁸³-Y⁵⁹⁷ segment for the membrane where it would sit on the surface with no defined secondary structure. A structural dependence of this segment on model membrane composition was observed. A helical conformation is favoured in detergent micelles and in the presence of negative charges. Our results suggest that the interaction of the S5-P linker with the membrane could participate in the stabilization of transient channel conformations, but helix formation would be triggered by interactions with other hERG domains. Because potential drug binding sites on the S5-P linker have been identified, we have explored the role of this segment in ALQTS. Four LQTS-liable drugs were studied which showed more affinity for the membrane than this hERG segment. Our results, therefore, identify two possible roles for the membrane in channel functioning and ALQTS.     

Mechanism of block of the hERG K+ channel by the scorpion toxin CnErg1

The scorpion toxin CnErg1 binds to human ether-a-go-go related gene (hERG) K(+) channels with a 1:1 stoichiometry and high affinity. However, in contrast to other scorpion toxin-ion channel interactions, the inhibition of macroscopic hERG currents by high concentrations of CnErg1 is incomplete. In this study, we have probed the molecular basis for this incomplete inhibition. High concentrations of CnErg1 had only modest effects on hERG gating that could not account for the incomplete block. Furthermore, the residual current in the presence of 1 microM CnErg1 had normal single channel conductance. Analysis of the kinetics of CnErg1 interaction with hERG indicated that CnErg1 binding is not diffusion-limited. A bimolecular binding scheme that incorporates an initial encounter complex and permits normal ion conduction was able to completely reproduce both the kinetics and steady-state level of CnErg1-hERG binding. This scheme provides a simple kinetic explanation for incomplete block; that is, relatively fast backward compared to forward rate constants for the interconversion of the toxin-channel encounter complex and the blocked toxin-channel complex. We have also examined the temperature-dependence of CnErg1 binding to hERG. The dissociation constant, K(d), for CnErg1 increases from 7.3 nM at 22 degrees C to 64 nM at 37 degrees C (i.e., the affinity decreases as temperature increases) and the proportion of binding events that lead to channel blockade decreases from 70% to 40% over the same temperature range. These temperature-dependent effects on CnErg1 binding correlate with a temperature-dependent decrease in the stability of the putative CnErg1 binding site, the amphipathic alpha-helix in the outer pore domain of hERG, assayed using circular dichroism spectropolarimetry. Collectively, our data provides a plausible kinetic explanation for incomplete blockade of hERG by CnErg1 that is consistent with the proposed highly dynamic conformation of the outer pore domain of hERG.     

Mapping the binding site of a human ether-a-go-go-related gene-specific peptide toxin (ErgTx) to the channel's outer vestibule

The goals of this study are to investigate the mechanism and site of action whereby a human ether-a-go-go-related gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteine-scanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx.Shaker interaction, elevating [K](o) (from 2 to 98 mm) does not affect ErgTx potency, and through-solution electrostatic forces only play a minor role in influencing ErgTx.HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (DeltaDeltaG > 2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic alpha-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx.HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule.     

Exploring structural features of the interaction between the scorpion toxinCnErg1 and ERG K+ channels

The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG channels on the turret region, while alpha-KTx3.2 Agitoxin-2 binds to the pore region of the Shaker K+ channel, and alpha-KTx5.3 BmP05 binds to the intermediate region of the small-conductance calcium-activated K-channel (SK(Ca)). In order to explore the critical residues for gamma-KTx binding, we determined the NMR structure of native gamma-KTx1.1 (CnErg1), a 42 amino acid residues scorpion toxin isolated from the venom of the Mexican scorpion Centruro?des noxius Hoffmann, and we used computational evolutionary trace (ET) analysis to predict possible structural and functional features of interacting surfaces. The 1H-NMR three-dimensional solution structure of native ergtoxin (CnErg1) was solved using a total of 452 distance constraints, 13 3J(NH-Halpha) and 10 hydrogen bonds. The structure is characterized by 2 segments of alpha-helices and a triple-stranded antiparallel beta-sheet stabilized by 4 disulfide bridges. The ET and structural analysis provided indication of the presence of two important amino acid residue clusters, one hydrophobic and the other hydrophilic, that should be involved in the surface contact between the toxin and the channel. Some features of the proposed interacting surface are discussed.     

Recombinant expression of the toxic peptide ErgTx1 and role of Met35 on its stability and function

Ergtoxin 1 (ErgTx1) is a 42 amino acid peptide purified from the venom of the Mexican scorpion Centruroides noxius Hoffmann, capable of blocking specifically human potassium channels of the ether-á-go-go-related gene family (hERG). This peptide binds to a partially overlapping site on the channel outer mouth, in which residues of the S5-P linker are critically involved. Here we describe results of site directed mutagenesis of the ErgTx1 gene and its heterologous expression in Escherichia coli. The recombinant products show the fundamental role played by methionine in position 35 (Met35) of the primary structure. Naturally oxidized Met35 decreases by three orders of magnitude the affinity of the peptide for the hERG1 channels. This result is quite relevant, because it shows two possible situations: either Met35 is involved in the proper folding of the molecule or it plays a direct role in the interaction with the channel, i.e., constitutes part of the interacting surfaces. These two situations were evaluated by preparing heterologously expressed ErgTx1 gene and a mutant containing alanine in position 35. Additionally circular dichroism measurements of both native and recombinant peptides were performed. The electrophysiological recordings and the structural values obtained by optical measurements, strongly support the idea that Met35 is indeed a key residue on the interacting surfaces of the toxin with the channels.     

Brownian dynamics simulations of interaction between scorpion toxin Lq2 and potassium ion channel

The association of the scorpion toxin Lq2 and a potassium ion (K(+)) channel has been studied using the Brownian dynamics (BD) simulation method. All of the 22 available structures of Lq2 in the Brookhaven Protein Data Bank (PDB) determined by NMR were considered during the simulation, which indicated that the conformation of Lq2 affects the binding between the two proteins significantly. Among the 22 structures of Lq2, only 4 structures dock in the binding site of the K(+) channel with a high probability and favorable electrostatic interactions. From the 4 candidates of the Lq2-K(+) channel binding models, we identified a good three-dimensional model of Lq2-K(+) channel complex through triplet contact analysis, electrostatic interaction energy estimation by BD simulation and structural refinement by molecular mechanics. Lq2 locates around the extracellular mouth of the K(+) channel and contacts the K(+) channel using its beta-sheet rather than its alpha-helix. Lys27, a conserved amino acid in the scorpion toxins, plugs the pore of the K(+) channel and forms three hydrogen bonds with the conserved residues Tyr78(A-C) and two hydrophobic contacts with Gly79 of the K(+) channel. In addition, eight hydrogen-bonds are formed between residues Arg25, Cys28, Lys31, Arg34 and Tyr36 of Lq2 and residues Pro55, Tyr78, Gly79, Asp80, and Tyr82 of K(+) channel. Many of them are formed by side chains of residues of Lq2 and backbone atoms of the K(+) channel. Thirteen hydrophobic contacts exist between residues Met29, Asn30, Lys31 and Tyr36 of Lq2 and residues Pro55, Ala58, Gly79, Asp80 and Tyr82 of the K(+) channel. These favorable interactions stabilize the association between the two proteins. These observations are in good agreement with the experimental results and can explain the binding phenomena between scorpion toxins and K(+) channels at the level of molecular structure. The consistency between the BD simulation and the experimental data indicates that our three-dimensional model of Lq2-K(+) channel complex is reasonable and can be used in further biological studies such as rational design of blocking agents of K(+) channels and mutagenesis in both toxins and K(+) channels.     

Proton block of the pore underlies the inhibition of hERG cardiac K+ channels during acidosis

Human ether-a-go-go-related gene (hERG) potassium channels are critical determinants of cardiac repolarization. Loss of function of hERG channels is associated with Long QT Syndrome, arrhythmia, and sudden death. Acidosis occurring as a result of myocardial ischemia inhibits hERG channel function and may cause a predisposition to arrhythmias. Acidic pH inhibits hERG channel maximal conductance and accelerates deactivation, likely by different mechanisms. The mechanism underlying the loss of conductance has not been demonstrated and is the focus of the present study. The data presented demonstrate that, unlike in other voltage-gated potassium (Kv) channels, substitution of individual histidine residues did not abolish the pH dependence of hERG channel conductance. Abolition of inactivation, by the mutation S620T, also did not affect the proton sensitivity of channel conductance. Instead, voltage-dependent channel inhibition (δ = 0.18) indicative of pore block was observed. Consistent with a fast block of the pore, hERG S620T single channel data showed an apparent reduction of the single channel current amplitude at low pH. Furthermore, the effect of protons was relieved by elevating external K(+) or Na(+) and could be modified by charge introduction within the outer pore. Taken together, these data strongly suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.     

The pore domain outer helix contributes to both activation and inactivation of the HERG K+ channel

Ion flow in many voltage-gated K(+) channels (VGK), including the (human ether-a-go-go-related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K(+) channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approximately every third residue, from Phe(551) to Trp(563), affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on activation (W563A, F559A, and F551A) face out toward the voltage sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala(561), His(562), Ala(565), Trp(568), and Ile(571)), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.     

ATP-sensitive potassium channels and vascular function

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker K_V channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian K_V1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of K_V1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth,¹ that is not seen in Shaker despite significant homology between it and Kv1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and K_V channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K⁺ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K⁺, and an IC₅₀ of 29 mM K⁺_o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K⁺, and further support the idea that outer pore motions occur rapidly after K_V1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.     

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating

Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.     

Effects of outer mouth mutations on hERG channel function: a comparison with similar mutations in the Shaker channel.

The fast-inactivation process in the hERG channel can be affected by mutations in the pore or S6 domain, similar to the C-type inactivation in the Shaker channel. However, differences in the kinetics and voltage dependence of inactivation between these two channels suggest that different structural determinants may be involved. To explore this possibility, we mutated a serine in the outer mouth region of hERG (S631) to residues of different physicochemical properties and compared the resulting changes in the channel's inactivation process with those resulting from mutations of an equivalent position in the Shaker channel (T449). The most dramatic differences are seen when this position is occupied by a charged residue: S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E facilitate C-type inactivation in Shaker. S631K and S631E also disrupted the K selectivity of hERG pore, a change not seen in T449K or T449E of Shaker. To further study why there are such differences, we replaced S631 with cysteine. This allowed us to manipulate the properties of thiol groups at position 631 and correlate side-chain properties here with changes in channel function. S631C behaved like the wild-type channel when the thiol groups were in the reduced state. Oxidizing thiol groups with H2O2 or modifying them with MTSET or MTSES disrupted C-type inactivation and K selectivity, similar to the phenotype of S631K and S631E. The same thiol-modifying maneuvers did not affect the wild-type channel function. Our results suggest differences in the outer mouth structure between hERG and Shaker, and we propose a "molecular spring" hypothesis to explain these differences.     

Molecular coupling in the human ether-a-go-go-related gene-1 (hERG1) K+ channel inactivation pathway

Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.