Open conformation of hERG channel turrets revealed by a specific scorpion toxin BmKKx2.
ABSTRACT: BACKGROUND: The human ether-a-go-go-related gene potassium channel (hERG) has an unusual long turret, whose role in recognizing scorpion toxins remains controversial. Here, BmKKx2, the first specific blocker of hERG channel derived from scorpion Mesobuthus martensii, was identified and the turret role of hERG channel was re-investigated using BmKKx2 as a molecular probe. RESULTS: BmKKx2 was found to block hERG channel with an IC50 of 6.7?±?1.7 nM and share similar functional surface with the known hERG channel inhibitor BeKm-1. The alanine-scanning mutagenesis data indicate that different residue substitutions on hERG channel by alanine decreased the affinities of toxin BmKKx2 by about 10-fold compared with that of wild-type hERG channel, which reveals that channel turrets play a secondary role in toxin binding. Different from channel turret, the pore region of hERG channel was found to exert the conserved and essential function for toxin binding because the mutant hERG-S631A channel remarkably decreased toxin BmKKx2 affinity by about 104-fold. CONCLUSIONS: Our results not only revealed that channel turrets of hERG channel formed an open conformation in scorpion toxin binding, but also enriched the diversity of structure-function relationships among the different potassium channel turrets.
Project description:Scorpion toxins are well-known as the largest potassium channel peptide blocker family. They have been successfully proven to be valuable molecular probes for structural research on diverse potassium channels. The potassium channel pore region, including the turret and filter regions, is the binding interface for scorpion toxins, and structural features from different potassium channels have been identified using different scorpion toxins. According to the spatial orientation of channel turrets with differential sequence lengths and identities, conformational changes and molecular surface properties, the potassium channel turrets can be divided into the following three states: open state with less hindering effects on toxin binding, half-open state or half-closed state with certain effects on toxin binding, and closed state with remarkable effects on toxin binding. In this review, we summarized the diverse structural features of potassium channels explored using scorpion toxin tools and discuss future work in the field of scorpion toxin-potassium channel interactions.
Project description:Peptidic toxins that target specifically mammalian channels and receptors can be found in the venom of animals. These toxins are rarely used directly as tools for biochemical experiments, and need to be modified via the attachment of chemical groups (e.g., radioactive or fluorescent moieties). Ideally, such modifications should maintain the toxin specificity and affinity for its target. With the goal of obtaining fluorescent derivatives of BeKm-1, a toxin from the scorpion species Buthus eupeus that selectively inhibits the voltage-gated potassium ion channel hERG, we produced four active analogues using a model of BeKm-1 docking to the outer mouth of the channel. In these BeKm-1 analogues, the natural peptide was linked to the fluorescent cyanine 5 (Cy5) probe via four different linkers at Arg1 or Arg/Lys27. All analogues retained their specificity towards the hERG channel in electrophysiological experiments but displayed a lesser affinity. These results validate our strategy for designing toxin analogues and demonstrate that different chemical groups can be attached to different residues of BeKm-1.
Project description:The 21-residue compact tertiapin-Q (TPNQ) toxin, a derivative of honey bee toxin tertiapin (TPN), is a potent blocker of inward-rectifier K(+) channel subtype, rat Kir1.1 (rKir1.1) channel, and their interaction mechanism remains unclear.Based on the flexible feature of potassium channel turrets, a good starting rKir1.1 channel structure was modeled for the accessibility of rKir1.1 channel turrets to TPNQ toxin. In combination with experimental alanine scanning mutagenesis data, computational approaches were further used to obtain a reasonable TPNQ toxin-rKir1.1 channel complex structure, which was completely different from the known binding modes between animal toxins and potassium channels. TPNQ toxin mainly adopted its helical domain as the channel-interacting surface together with His12 as the pore-blocking residue. The important Gln13 residue mainly contacted channel residues near the selectivity filter, and Lys20 residue was surrounded by a polar "groove" formed by Arg118, Thr119, Glu123, and Asn124 in the channel turret. On the other hand, four turrets of rKir1.1 channel gathered to form a narrow pore entryway for TPNQ toxin recognition. The Phe146 and Phe148 residues in the channel pore region formed strong hydrophobic protrusions, and produced dominant nonpolar interactions with toxin residues. These specific structure features of rKir1.1 channel vestibule well matched the binding of potent TPNQ toxin, and likely restricted the binding of the classical animal toxins.The TPNQ toxin-rKir1.1 channel complex structure not only revealed their unique interaction mechanism, but also would highlight the diverse animal toxin-potassium channel interactions, and elucidate the relative insensitivity of rKir1.1 channel towards animal toxins.
Project description:<h4>Background</h4>The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.<h4>Principal findings</h4>The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC50 of 6.7 ± 1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca(2+) concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process.<h4>Conclusions/significance</h4>Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.
Project description:Although many studies concerning the sensitivity mechanism of scorpion toxin-potassium channel interactions have been reported, few have explored the biochemical insensitivity mechanisms of potassium channel receptors toward natural scorpion toxin peptides, such as the KCNQ1 channel. Here, by sequence alignment analyses of the human KCNQ1 channel and scorpion potassium channel MmKv2, which is completely insensitive to scorpion toxins, we proposed that the insensitivity mechanism of KCNQ1 toward natural scorpion toxins might involve two functional regions, the turret and filter regions. Based on this observation, a series of KCNQ1 mutants were constructed to study molecular mechanisms of the KCNQ1 channel insensitivity toward natural scorpion toxins. Electrophysiological studies of chimera channels showed that the channel filter region controls KCNQ1 insensitivity toward the classical scorpion toxin ChTX. Interestingly, further residue mutant experiments showed that a single basic residue in the filter region determined the insensitivity of KCNQ1 channels toward scorpion toxins. Our present work showed that amino acid residue diversification at common sites controls the sensitivity and insensitivity of potassium channels toward scorpion toxins. The unique insensitivity mechanism of KCNQ1 toward natural scorpion toxins will accelerate the rational design of potent peptide inhibitors toward this channel.
Project description:The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Dysfunction of hERG causes long QT syndrome and sudden death, which occur in patients with cardiac ischemia. Cardiac ischemia is also associated with activation, up-regulation, and secretion of various proteolytic enzymes. Here, using whole-cell patch clamp and Western blotting analysis, we demonstrate that the hERG/IKr channel was selectively cleaved by the serine protease, proteinase K (PK). Using molecular biology techniques including making a chimeric channel between protease-sensitive hERG and insensitive human ether-a-go-go (hEAG), as well as application of the scorpion toxin BeKm-1, we identified that the S5-pore linker of hERG is the target domain for proteinase K cleavage. To investigate the physiological relevance of the unique susceptibility of hERG to proteases, we show that cardiac ischemia in a rabbit model was associated with a reduction in mature ERG expression and an increase in the expression of several proteases, including calpain. Using cell biology approaches, we found that calpain-1 was actively released into the extracellular milieu and cleaved hERG at the S5-pore linker. Using protease cleavage-predicting software and site-directed mutagenesis, we identified that calpain-1 cleaves hERG at position Gly-603 in the S5-pore linker of hERG. Clarification of protease-mediated damage of hERG extends our understanding of hERG regulation. Damage of hERG mediated by proteases such as calpain may contribute to ischemia-associated QT prolongation and sudden cardiac death.
Project description:IKr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go-Related Gene (hERG, Kv11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.
Project description:The hERG potassium channel is critical to normal repolarization of cardiac action potentials (APs) and loss- and gain-of-function hERG mutations are associated, respectively, with long and short QT syndromes, pathological conditions that can lead to arrhythmias and sudden death. hERG current (IhERG ) exhibits uniquely fast inactivation involving conformational changes to the channel pore. The S631A hERG pore mutation was originally engineered to interrogate hERG channel inactivation, but has very recently been found in a family with short QT syndrome (SQTS). Accordingly, this study characterized the effects of the S631A mutation on IhERG profile during ventricular, atrial, and Purkinje fiber (PF) AP waveforms, using patch clamp recording from hERG expressing HEK 293 cells at 37°C. Under conventional voltage clamp, the current-voltage (I-V) relation for IhERG exhibited a marked right-ward shift in the region of negative slope at positive membrane potentials. Under ventricular AP clamp, the S631A mutation resulted in augmented IhERG , which also peaked much earlier during the AP plateau than did wild-type (WT) IhERG . Instantaneous I-V relations showed a marked positive shift in peak repolarizing current during the ventricular AP in the S631A setting, while the instantaneous conductance-voltage relation showed an earlier and more sustained rise in S631A compared to WT IhERG conductance during ventricular repolarization. Experiments with atrial and PF APs in each case also showed augmented and positively shifted IhERG in the S631A setting, indicating that the S631A mutation is likely to accelerate repolarization in all three cardiac regions. Ventricular AP clamp experiments showed retained effectiveness of the class Ia antiarrhythmic drug quinidine (1 ?mol/L) against S631A IhERG . Quinidine is thus likely to be effective in reducing excessively fast repolarization in SQTS resulting from the S631A hERG mutation.
Project description:Studies in Shaker, a voltage-dependent potassium channel, suggest a coupling between activation and inactivation. This coupling is controversial in hERG, a fast-inactivating voltage-dependent potassium channel. To address this question, we transferred to hERG the S3-S4 linker of the voltage-independent channel, rolf, to selectively disrupt the activation process. This chimera shows an intact voltage-dependent inactivation process consistent with a weak coupling, if any, between both processes. Kinetic models suggest that the chimera presents only an open and an inactivated states, with identical transition rates as in hERG. The lower sensitivity of the chimera to BeKm-1, a hERG preferential closed-state inhibitor, also suggests that the chimera presents mainly open and inactivated conformations. This chimera allows determining the mechanism of action of hERG blockers, as exemplified by the test on ketoconazole.
Project description:The short QT syndrome (SQTS) is associated with cardiac arrhythmias and sudden death. The SQT1 form of SQTS results from an inactivation-attenuated, gain-of-function mutation (N588K) to the human ether-à-go-go-related gene (hERG) potassium channel. Pharmacological blockade of this mutated hERG channel may have therapeutic value. However, hERG-blocking potencies of canonical inhibitors such as E-4031 and D-sotalol are significantly reduced for N588K-hERG. Here, five hERG-blocking drugs were compared to determine their relative potencies for inhibiting N588K channels, and two other inactivation-attenuated mutant channels were tested to investigate the association between impaired inactivation and altered drug potency.Whole-cell patch clamp measurements of hERG current (I(hERG)) mediated by wild-type and mutant (N588K, S631A and N588K/S631A) channels were made at 37 degrees C CHO cells.The N588K mutation attenuated I(hERG) inhibition in the following order: E-4031>amiodarone>quinidine>propafenone>disopyramide. Comparing the three inactivation mutants, the two single mutations, although occurring in different modules of the channel, attenuated inactivation to a nearly identical degree, whereas the double mutant caused considerably greater attenuation, permitting the titration of inactivation. Attenuation of channel inhibition was similar between the single mutants for each drug, and was significantly greater with the double mutant.The degree of drug inhibition of hERG channels may vary based on the level of channel inactivation. Drugs previously identified as useful for treating SQT1 have the least dependence on hERG inactivation. In addition, our findings indicate that amiodarone may warrant further investigation as a potential treatment for SQT1.