S5-P Linker Research Articles

Hydrophobic interactions between the voltage sensor and pore mediate inactivation in Kv11.1 channels

Kv11.1 channels are critical for the maintenance of a normal heart rhythm. The flow of potassium ions through these channels is controlled by two voltage-regulated gates, termed “activation” and “inactivation,” located at opposite ends of the pore. Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating. Although it is clear that the fourth transmembrane segments (S4), within each subunit of the tetrameric channel, are important for controlling the opening and closing of the activation gate, their role during inactivation gating is much less clear. Here, we use rate equilibrium free energy relationship (REFER) analysis to probe the contribution of the S4 “voltage-sensor” helix during inactivation of Kv11.1 channels. Contrary to the important role that charged residues play during activation gating, it is the hydrophobic residues (Leu529, Leu530, Leu532, and Val535) that are the key molecular determinants of inactivation gating. Within the context of an interconnected multi-domain model of Kv11.1 inactivation gating, our REFER analysis indicates that the S4 helix and the S4–S5 linker undergo a conformational rearrangement shortly after that of the S5 helix and S5P linker, but before the S6 helix. Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices. Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

Functional characterization of the LQT2-causing mutation R582C and the associated voltage-dependent fluorescence signal

The R582C mutation is one of many Long-QT Syndrome type 2 (LQT2)-causing mutations localized to the human ether-a-go-go related gene (hERG) channel's S5-P linker subdomain, yet its specific mechanism of dysfunction has not been examined. This study sought to characterize the biophysical properties of the congenital LQT2-causing mutation, R582C, and utilize this mutation to provide the first report of voltage-dependent fluorescence from the S5-P linker. Properties of the R582C channels were characterized by heterologous expression in both HEK293 cells and Xenopus oocytes using a combination of patch-clamp, 2-electrode voltage-clamp, immunoblot assay, and voltage-clamp fluorimetry. Expression of hERG R582C was found to be deficient in HEK293 cells, yet was amenable to rescue by incubation at reduced temperature or by treatment with dofetilide. Rescued channels expressed at levels comparable to wild type (WT) channels. Kinetic differences result in decreased outward repolarizing current evoked by an action potential clamp protocol. Voltage-clamp fluorimetry experiments utilized the introduced cysteine to covalently attach a fluorescent probe (tetramethylrhodamine-5-maleimide) to the S5-P linker to directly observe conformational changes occurring due to inactivation. The major mechanism underlying pathogenicity of the R582C mutation is a trafficking deficiency, although channels also exhibit kinetic deficiencies, perhaps reflecting the position of the mutation in the pore turret. Voltage clamp fluorescence signals from R582C channels provide evidence that the hERG turret undergoes distinct conformational changes during inactivation.

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.

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 I 583–Y 597 with bicelles. Liquid and solid-state NMR experiments as well as circular dichroism results show a strong affinity of the I 583–Y 597 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.

Extracellular K+ Removal Leads to a Complete Conductance Loss that Triggers Internalization of the Cell Surface hERG Channels

Potassium channels are present in a wide variety of cells and play important roles in cell functions. Although the gating properties of potassium channels have been extensively studied, it is not known whether and how functional states of a channel affect the channel's membrane stability. The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunits of the rapidly activating delayed rectifier potassium channel (IKr) that is important for cardiac repolarization. Here, we demonstrate that a reduction in [K+]o decreased IhERG in a concentration dependent manner, and exposure of cells to 0 mM K+o completely eliminated hERG conductance within 3 min. Notably, the conductance-lost channels due to 0 mM K+ exposure could not be readily reversed to the functional state upon re-exposure to normal MEM (5 mM K+), and they were totally internalized within 4 h under 0 mM K+o culture conditions. The hERG-permeable cations ion Rb+ or Cs+ (5 mM) effectively prevented both hERG conductance loss and internalization caused by 0 mM K+o exposure. Point mutations in hERG pore helix and selectivity filter such as the S624T and F627Y, but not in the S5-P linker and S6 regions, eradicated both 0 mM K+o induced conductance-loss and internalization of hERG channels. Upon exposure to 0 mM K+ medium, WT hERG channels, but not the S624T mutant channels, colocalized with ubiquitin. Overexpression of ubiquitin enhanced degradation of the mature form of WT, but not the S624T mutant hERG channels under 0 mM K+o conditions. Our data demonstrate that the presence of K+o is a prerequisite for hERG channel function, and the K+o-dependent functional state determines the hERG channel membrane stability.

State-Dependent Accessibility of the P-S6 Linker of Pacemaker (HCN) Channels Supports a Dynamic Pore-to-Gate Coupling Model

The hyperpolarization-activated cyclic nucleotide-modulated channel gene family (HCN1-4) encodes the membrane depolarizing current that underlies pacemaking. Although the topology of HCN resembles Kv channels, much less is known about their structure-function correlation. Previously, we identified several pore residues in the S5-P linker and P-loop that are externally accessible and/or influence HCN gating, and proposed an evolutionarily conserved pore-to-gate mechanism. Here we sought dynamic evidence by assessing the functional consequences of Cys-scanning substitutions in the unexplored P-S6 linker (residues 352–359), the HCN1-R background (that is, resistant to sulfhydryl-reactive agents). None of A352C, Q353C, A354C, P355C, V356C, S357C, M358C, or S359C produced functional currents; the loss-of-function of Q353C, A354C, S357C, and M358C could be rescued by the reducing agent dithiothreitol. Q353C, A354C, and S357C, but not M358C and HCN1-R, were sensitive to Cd2+ blockade (IC50 = 3–12 μM vs. >1 mM). External application of the positively charged covalent sulfhydryl modifier MTSET irreversibly reduced I−140mV of Q353C and A354C to 27.9 ± 3.4% and 58.2 ± 13.1% of the control, respectively, and caused significant steady-state activation shifts (∆V1/2 = –21.1 ± 1.6 for Q353C and −10.0 ± 2.9 mV for A354C). Interestingly, MTSET reactivity was also state dependent. MTSET, however, affected neither S357C nor M358C, indicating site specificity. Collectively, we have identified novel P-S6 residues whose extracellular accessibility was sterically and state dependent and have provided the first functional evidence consistent with a dynamic HCN pore-to-gate model.

The Kv1.2 potassium channel: The position of an N-glycan on the extracellular linkers affects its protein expression and function

Voltage-gated potassium Kv1 channels have three extracellular linkers, the S1–S2, the S3–S4, and the S5-P. The S1–S2 is the only linker that has an N-glycan and it is at a conserved position on this linker on Kv1.1–Kv1.5 and Kv1.7 channels. We hypothesize that an N-glycan is found at only this position due to its effect on folding, trafficking, and/or function of these channels. To investigate this hypothesis, N-glycosylation sites were engineered at different positions on the extracellular linkers of Kv1.2 to determine the effects of N-glycans on channel surface protein expression and function. Our data suggest that for Kv1 channels, (1) placing an N-glycan at non-native positions on the S1–S2 linker decreased cell surface protein expression but the N-glycan still affected function similarly as if it were at its native position, (2) placing a non-native N-glycan on the S3–S4 linker significantly altered function, and (3) placing a non-native N-glycan on the S5-P linker disrupted both trafficking and function. We suggest that Kv1 channels have an N-glycan at a conserved position on only the S1–S2 linker to overcome the constraints for proper folding, trafficking, and function that appear to occur if the N-glycan is moved from this position.

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.

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.

A Direct Demonstration of Closed-State Inactivation of K+ Channels at Low pH

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K+ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.

Molecular Dynamics and Continuum Electrostatics Studies of Inactivation in the HERG Potassium Channel

Fast inactivation of the HERG potassium channel plays a critical role in normal cardiac function. Malfunction of these channels due to either genetic mutations or blockade by drugs leads to cardiac arrhythmias. An unusually long S5-P linker in the outer mouth of HERG is implicated in the fast inactivation mechanism. To examine the role of the S5-P linker in this inactivation mechanism, we study the permeation properties of the open and inactive states of a recent homology model of HERG. This model was constructed using the KcsA potassium channel as a template and contains specific conformations of the S5-P linker in the open and inactive states. We perform molecular dynamics simulations on the HERG model, followed by free energy, structural, and continuum electrostatics calculations. Our free energy calculations lead to selectivity results of the model channel (K+ over Na+) that are different in some respects from those of other potassium channels but consistent with experimental observations. Our structural results show that, in the inactive state, the S5-P linkers move closer to the channel axis, possibly causing a steric hindrance to permeating K+ ions. Our electrostatics calculations reveal, in the inactive state, an electrostatic potential energy barrier of approximately 14 kT at the extracellular pore entrance, again sufficient to stop K+ ion permeation through the pore. These results suggest that a steric and/or electrostatic plug mechanism contributes to inactivation in the HERG homology model.

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.

Linkage between ‘disruption of inactivation’ and ‘reduction of K+ selectivity’ among hERG mutants in the S5‐P linker region

Linkage between ‘disruption of inactivation’ and ‘reduction of K⁺ selectivity’ among hERG mutants in the S5‐P linker region

SCAM analysis reveals a discrete region of the pore turret that modulates slow inactivation in Kv1.5

In Kv1.5, protonation of histidine 463 in the S5-P linker (turret) increases the rate of depolarization-induced inactivation and decreases the peak current amplitude. In this study, we examined how amino acid substitutions that altered the physico-chemical properties of the side chain at position 463 affected slow inactivation and then used the substituted cysteine accessibility method (SCAM) to probe the turret region (E456-P468) to determine whether residue 463 was unique in its ability to modulate the macroscopic current. Substitutions at position 463 of small, neutral (H463G and H463A) or large, charged (H463R, H463K, and H463E) side groups accelerated inactivation and induced a dependency of the current amplitude on the external potassium concentration. When cysteine substitutions were made in the distal turret (T462C-P468C), modification with either the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) or negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate reagent irreversibly inhibited current. This inhibition could be antagonized either by the R487V mutation (homologous to T449V in Shaker) or by raising the external potassium concentration, suggesting that current inhibition by MTS reagents resulted from an enhancement of inactivation. These results imply that protonation of residue 463 does not modulate inactivation solely by an electrostatic interaction with residues near the pore mouth, as proposed by others, and that residue 463 is part of a group of residues within the Kv1.5 turret that can modulate P/C-type inactivation.

Effect of S5P α‐helix charge mutants on inactivation of hERG K+ channels

The ether-à-go-go (EAG) family of voltage-gated K(+) channels contains three subfamilies, EAG, ether-à-go-go related (ERG) and ether-à-go-go like (ELK). The human ether-à-go-go related gene (hERG) K(+) channel has been of significant interest because loss of function in the hERG channel is associated with a markedly increased risk of cardiac arrhythmias. The hERG channel has unusual kinetics with slow activation and deactivation but very rapid and voltage-dependent inactivation. The outer pore region of the hERG K(+) channel is predicted to be different from that of other members of the voltage-gated K(+) channel family. HERG has a much longer linker between the fifth transmembrane domain (SS) and the pore helix (S5P linker) compared to other families of voltage-gated K(+) channels (43 amino acids compared to 14-23 amino acids). Further, the S5P linker contains an amphipathic alpha-helix that in hERG channels probably interacts with the mouth of the pore to modulate inactivation. The human EAG and rat ELK2 channels (hEAG and rELK2) show reduced or no inactivation in comparison to hERG channels, yet both channels are predicted to contain a similarly long S5P linker to that of hERG. In this study, we have constructed a series of chimaeric channels consisting of the S1-S6 of hERG but with the S5P alpha-helical region of either hEAG or rELK2, and one consisting of the S1-S6 of rELK2 but with the S5P alpha-helical region of hERG to investigate the role of the S5P linker in inactivation. Our studies show that charged residues on the alpha-helix of the S5P linker contribute significantly to the differences in inactivation characteristics of the EAG family channels. Further, individually mutating each of the hydrophilic residues on the S5P alpha-helix of hERG to a charged residue had significant effects on the voltage dependence of inactivation and the two residues with the greatest affect when mutated to a lysine, N588 and Q592, both lie on the same face of the S5P alpha -helix. We suggest that inactivation of hERG involves the interaction of this face of the S5P alpha-helix with a charged residue on the remainder of the outer pore domain of the channel.

Dynamic conformational changes of extracellular S5–P linkers in the hERG channel

The hERG channel has an unusually long 'S5-P linker' (residues 571-613) that lines the outer mouth of the pore. Previously, we have shown that residues along this S5-P linker are critical for the fast-inactivation process and K(+) selectivity of the hERG channel. Here we used several approaches to probe the structure of this S5-P linker and its interactions with other domains of the hERG channel. Circular dichroism and NMR analysis of a synthetic hERG S5-P linker peptide suggested that this linker is quite dynamic: its central region (positions 583-593) can be unstructured or helical, depending on whether it is immersed in an aqueous phase or in contact with a hydrophobic environment. Cysteine introduced into positions 583-597 of the S5-P linker can form intersubunit disulphide bonds, and at least four of them (at 584, 585, 588 and 589) can form disulphide bonds with counterparts from neighbouring subunits. We propose that the four S5-P linkers in a hERG channel can engage in dynamic conformational changes during channel gating, and interactions between S5-P linkers from neighbouring subunits contribute importantly to channel inactivation.

The N588K-HERG K + channel mutation in the ‘short QT syndrome’: Mechanism of gain-in-function determined at 37 °C

The idiopathic short QT syndrome (SQTS) is characterised by an abnormally short QT interval on the electrocardiogram and by an increased risk of arrhythmia and sudden death. One variant of the syndrome is linked to missense mutations that lead to a single amino-acid change (N588K; asparagine to lysine) in the S5-Pore linker region of the cardiac HERG K + channel. This study was performed in order to determine how the N588K mutation alters HERG channel current ( I HERG) kinetics at mammalian physiological temperature. The whole-cell current–voltage ( I– V) relation for wild-type (WT) I HERG measured from Chinese Hamster Ovary cells was maximal at ∼0 mV and showed marked inward rectification positive to this. In contrast, N588K I HERG showed marked rectification only at +60 mV and at more positive voltages. The voltage dependence of activation of N588K-HERG did not differ significantly from that of WT-HERG. However, N588K I HERG had a significantly more positive inactivation V 0.5 (−8.14 ± 0.82 mV) than did WT I HERG (−70.05 ± 0.82 mV; P < 0.001, unpaired t test; n = 5 for each). Its P Na/ P K ratio was also greater. The instantaneous I– V relation for N588K I HERG under action potential voltage clamp peaked at ∼+40 mV, compared to ∼−37 mV for WT- I HERG. These findings underscore the importance of the S5-P linker in HERG channel function and indicate that N588K-HERG contributes increased repolarising current earlier in the ventricular action potential at physiological temperature due to a ∼+60 mV shift in voltage dependence of I HERG inactivation.

Unique interaction of scorpion toxins with the hERG channel.

ERG potassium channels specify one component of the delayed rectifier in the heart and are likely to play an important functional role in other excitable cells. Compared to other K+ channels, the human ERG (hERG) channel possesses an unusually long S5-P linker that presumably forms an alpha-helix important for channel function. hERG-specific toxins bind to the outer mouth of the hERG channel. Channel residues in the middle of the S5-P linker and at the pore entrance are critical for toxin binding. One of these scorpion toxins is BeKm-1. Residues critical for BeKm-1 binding to the hERG channel are located in the alpha-helix and the following loop, whereas the "traditional" interaction surface of other short scorpion toxins is formed by residues on the beta-sheet. This unique localization of BeKm-1's interaction surface and its specific action on the hERG channel suggest a unique outer mouth structure of the hERG channel. We used the mutant cycle analysis approach to define contacts in the toxin-channel complex. This information provides critical constraints and is important for molecular modeling of the hERG pore structure.

A Superfamily of Voltage-gated Sodium Channels in Bacteria

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.

Structure of the HERG K+ Channel S5P Extracellular Linker: ROLE OF AN AMPHIPATHIC α-HELIX IN C-TYPE INACTIVATION

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585-Ile593 and Gly604-Tyr611 of the channel. The Trp585-Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604-Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.