In silico models of the macromolecular NaV1.5-KIR2.1 complex.

In cardiac cells, the expression of the cardiac voltage-gated Na+ channel (NaV1.5) is reciprocally regulated with the inward rectifying K+ channel (KIR2.1). These channels can form macromolecular complexes that pre-assemble early during forward trafficking (transport to the cell membrane). In this study, we present in silico 3D models of NaV1.5-KIR2.1, generated by rigid-body protein-protein docking programs and deep learning-based AlphaFold-Multimer software. Modeling revealed that the two channels could physically interact with each other along the entire transmembrane region. Structural mapping of disease-associated mutations revealed a hotspot at this interface with several trafficking-deficient variants in close proximity. Thus, examining the role of disease-causing variants is important not only in isolated channels but also in the context of macromolecular complexes. These findings may contribute to a better understanding of the life-threatening cardiovascular diseases underlying KIR2.1 and NaV1.5 malfunctions.

Subunit gating resulting from individual protonation events in Kir2 channels.

Inwardly rectifying potassium (Kir) channels open at the 'helix bundle crossing' (HBC), formed by the M2 helices at the cytoplasmic end of the transmembrane pore. Introduced negative charges at the HBC (G178D) in Kir2.2 channels forces opening, allowing pore wetting and free movement of permeant ions between the cytoplasm and the inner cavity. Single-channel recordings reveal striking, pH-dependent, subconductance behaviors in G178D (or G178E and equivalent Kir2.1[G177E]) mutant channels, with well-resolved non-cooperative subconductance levels. Decreasing cytoplasmic pH shifts the probability towards lower conductance levels. Molecular dynamics simulations show how protonation of Kir2.2[G178D], or the D173 pore-lining residues, changes solvation, K+ ion occupancy, and K+ conductance. Ion channel gating and conductance are classically understood as separate processes. The present data reveal how individual protonation events change the electrostatic microenvironment of the pore, resulting in step-wise alterations of ion pooling, and hence conductance, that appear as 'gated' substates.

Binding of RPR260243 at the intracellular side of the hERG1 channel pore domain slows closure of the helix bundle crossing gate.

The opening and closing of voltage-dependent potassium channels is dependent on a tight coupling between movement of the voltage sensing S4 segments and the activation gate. A specific interaction between intracellular amino- and carboxyl-termini is required for the characteristically slow rate of channel closure (deactivation) of hERG1 channels. Compounds that increase hERG1 channel currents represent a novel approach for prevention of arrhythmia associated with prolonged ventricular repolarization. RPR260243 (RPR), a quinoline oxo-propyl piperidine derivative, inhibits inactivation and dramatically slows the rate of hERG1 channel deactivation. Here we report that similar to its effect on wild-type channels, RPR greatly slows the deactivation rate of hERG1 channels missing their amino-termini, or of split channels lacking a covalent link between the voltage sensor domain and the pore domain. By contrast, RPR did not slow deactivation of C-terminal truncated hERG1 channels or D540K hERG1 mutant channels activated by hyperpolarization. Together, these findings indicate that ability of RPR to slow deactivation requires an intact C-terminus, does not slow deactivation by stabilizing an interaction involving the amino-terminus or require a covalent link between the voltage sensor and pore domains. All-atom molecular dynamics simulations using the cryo-EM structure of the hERG1 channel revealed that RPR binds to a pocket located at the intracellular ends of helices S5 and S6 of a single subunit. The slowing of channel deactivation by RPR may be mediated by disruption of normal S5-S6 interactions.

Subunit gating resulting from individual protonation events in Kir2 channels.

Inwardly rectifying potassium (Kir) channels play a critical role in stabilizing the membrane potential, thus controlling numerous physiological phenomena in multiple tissues. Channel conductance is activated by cytoplasmic modulators that open the channel at the 'helix bundle crossing' (HBC), formed by the coming together of the M2 helices from each of the four subunits, at the cytoplasmic end of the transmembrane pore. We introduced a negative charge at the bundle crossing region (G178D) in classical inward rectifier Kir2.2 channel subunits that forces channel opening, allowing pore wetting and free movement of permeant ions between the cytoplasm and the inner cavity. Single-channel recordings reveal a striking pH-dependent subconductance behavior in G178D (or G178E and equivalent Kir2.1[G177E]) mutant channels that reflects individual subunit events. These subconductance levels are well resolved temporally and occur independently, with no evidence of cooperativity. Decreasing cytoplasmic pH shifts the probability towards lower conductance levels, and molecular dynamics simulations show how protonation of Kir2.2[G178D] and, additionally, the rectification controller (D173) pore-lining residues leads to changes in pore solvation, K+ ion occupancy, and ultimately K+ conductance. While subconductance gating has long been discussed, resolution and explanation have been lacking. The present data reveals how individual protonation events change the electrostatic microenvironment of the pore, resulting in distinct, uncoordinated, and relatively long-lasting conductance states, which depend on levels of ion pooling in the pore and the maintenance of pore wetting. Gating and conductance are classically understood as separate processes in ion channels. The remarkable sub-state gating behavior of these channels reveals how intimately connected 'gating' and 'conductance' are in reality.

ß subunits of GABAA receptors form proton-gated chloride channels: Insights into the molecular basis.

Gamma-aminobutyric acid type A receptors (GABAARs) are ligand gated channels mediating inhibition in the central nervous system. Here, we identify a so far undescribed function of ß-subunit homomers as proton-gated anion channels. Mutation of a single H267A in ß3 subunits completely abolishes channel activation by protons. In molecular dynamic simulations of the ß3 crystal structure protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit leading to a pore stabilising ring formation and accumulation of Cl- within the transmembrane pore. Conversion of these residues in proton insensitive ρ1 subunits transfers proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH changes (pH50 is in the range 6- 6.3) and kinetic studies suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus as potential novel drug target.

The Bradycardic Agent Ivabradine Acts as an Atypical Inhibitor of Voltage-Gated Sodium Channels.

Background and purpose: Ivabradine is clinically administered to lower the heart rate, proposedly by inhibiting hyperpolarization-activated cyclic nucleotide-gated cation channels in the sinoatrial node. Recent evidence suggests that voltage-gated sodium channels (VGSC) are inhibited within the same concentration range. VGSCs are expressed within the sinoatrial node and throughout the conduction system of the heart. A block of these channels thus likely contributes to the established and newly raised clinical indications of ivabradine. We, therefore, investigated the pharmacological action of ivabradine on VGSCs in sufficient detail in order to gain a better understanding of the pro- and anti-arrhythmic effects associated with the administration of this drug. Experimental Approach: Ivabradine was tested on VGSCs in native cardiomyocytes isolated from mouse ventricles and the His-Purkinje system and on human Nav1.5 in a heterologous expression system. We investigated the mechanism of channel inhibition by determining its voltage-, frequency-, state-, and temperature-dependence, complemented by a molecular drug docking to the recent Nav1.5 cryoEM structure. Automated patch-clamp experiments were used to investigate ivabradine-mediated changes in Nav1.5 inactivation parameters and inhibition of different VGSC isoforms. Key results: Ivabradine inhibited VGSCs in a voltage- and frequency-dependent manner, but did not alter voltage-dependence of activation and fast inactivation, nor recovery from fast inactivation. Cardiac (Nav1.5), neuronal (Nav1.2), and skeletal muscle (Nav1.4) VGSC isoforms were inhibited by ivabradine within the same concentration range, as were sodium currents in native cardiomyocytes isolated from the ventricles and the His-Purkinje system. Molecular drug docking suggested an interaction of ivabradine with the classical local anesthetic binding site. Conclusion and Implications: Ivabradine acts as an atypical inhibitor of VGSCs. Inhibition of VGSCs likely contributes to the heart rate lowering effect of ivabradine, in particular at higher stimulation frequencies and depolarized membrane potentials, and to the observed slowing of intra-cardiac conduction. Inhibition of VGSCs in native cardiomyocytes and across channel isoforms may provide a potential basis for the anti-arrhythmic potential as observed upon administration of ivabradine.

A selectivity filter mutation provides insights into gating regulation of a K+ channel.

G-protein coupled inwardly rectifying potassium (GIRK) channels are key players in inhibitory neurotransmission in heart and brain. We conducted molecular dynamics simulations to investigate the effect of a selectivity filter (SF) mutation, G154S, on GIRK2 structure and function. We observe mutation-induced loss of selectivity, changes in ion occupancy and altered filter geometry. Unexpectedly, we reveal aberrant SF dynamics in the mutant to be correlated with motions in the binding site of the channel activator Gßγ. This coupling is corroborated by electrophysiological experiments, revealing that GIRK2wt activation by Gßγ reduces the affinity of Ba2+ block. We further present a functional characterization of the human GIRK2G154S mutant validating our computational findings. This study identifies an allosteric connection between the SF and a crucial activator binding site. This allosteric gating mechanism may also apply to other potassium channels that are modulated by accessory proteins.

Simulating PIP2-Induced Gating Transitions in Kir6.2 Channels.

ATP-sensitive potassium (KATP) channels consist of an inwardly rectifying K+ channel (Kir6.2) pore, to which four ATP-sensitive sulfonylurea receptor (SUR) domains are attached, thereby coupling K+ permeation directly to the metabolic state of the cell. Dysfunction is linked to neonatal diabetes and other diseases. K+ flux through these channels is controlled by conformational changes in the helix bundle region, which acts as a physical barrier for K+ permeation. In addition, the G-loop, located in the cytoplasmic domain, and the selectivity filter might contribute to gating, as suggested by different disease-causing mutations. Gating of Kir channels is regulated by different ligands, like Gßγ, H+, Na+, adenosine nucleotides, and the signaling lipid phosphatidyl-inositol 4,5-bisphosphate (PIP2), which is an essential activator for all eukaryotic Kir family members. Although molecular determinants of PIP2 activation of KATP channels have been investigated in functional studies, structural information of the binding site is still lacking as PIP2 could not be resolved in Kir6.2 cryo-EM structures. In this study, we used Molecular Dynamics (MD) simulations to examine the dynamics of residues associated with gating in Kir6.2. By combining this structural information with functional data, we investigated the mechanism underlying Kir6.2 channel regulation by PIP2.

Development of IKATP Ion Channel Blockers Targeting Sulfonylurea Resistant Mutant KIR6.2 Based Channels for Treating DEND Syndrome.

Introduction: DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes. Gain of function mutations in the KCNJ11 gene, encoding the KIR6.2 subunit of the IKATP potassium channel, stand at the basis of most forms of DEND syndrome. In a previous search for existing drugs with the potential of targeting Cantú Syndrome, also resulting from increased IKATP, we found a set of candidate drugs that may also possess the potential to target DEND syndrome. In the current work, we combined Molecular Modelling including Molecular Dynamics simulations, with single cell patch clamp electrophysiology, in order to test the effect of selected drug candidates on the KIR6.2 WT and DEND mutant channels. Methods: Molecular dynamics simulations were performed to investigate potential drug binding sites. To conduct in vitro studies, KIR6.2 Q52R and L164P mutants were constructed. Inside/out patch clamp electrophysiology on transiently transfected HEK293T cells was performed for establishing drug-channel inhibition relationships. Results: Molecular Dynamics simulations provided insight in potential channel interaction and shed light on possible mechanisms of action of the tested drug candidates. Effective IKIR6.2/SUR2a inhibition was obtained with the pore-blocker betaxolol (IC50 values 27-37 µM). Levobetaxolol effectively inhibited WT and L164P (IC50 values 22 µM) and Q52R (IC50 55 µM) channels. Of the SUR binding prostaglandin series, travoprost was found to be the best blocker of WT and L164P channels (IC50 2-3 µM), while Q52R inhibition was 15-20% at 10 µM. Conclusion: Our combination of MD and inside-out electrophysiology provides the rationale for drug mediated IKATP inhibition, and will be the basis for 1) screening of additional existing drugs for repurposing to address DEND syndrome, and 2) rationalized medicinal chemistry to improve IKATP inhibitor efficacy and specificity.

Computational Insights Into Voltage Dependence of Polyamine Block in a Strong Inwardly Rectifying K+ Channel.

Inwardly rectifying potassium (KIR) channels play important roles in controlling cellular excitability and K+ ion homeostasis. Under physiological conditions, KIR channels allow large K+ influx at potentials negative to the equilibrium potential of K+ but permit little outward current at potentials positive to the equilibrium potential of K+, due to voltage dependent block of outward K+ flux by cytoplasmic polyamines. These polycationic molecules enter the KIR channel pore from the intracellular side. They block K+ ion movement through the channel at depolarized potentials, thereby ensuring, for instance, the long plateau phase of the cardiac action potential. Key questions concerning how deeply these charged molecules migrate into the pore and how the steep voltage dependence arises remain unclear. Recent MD simulations on GIRK2 (=Kir3.2) crystal structures have provided unprecedented details concerning the conduction mechanism of a KIR channel. Here, we use MD simulations with applied field to provide detailed insights into voltage dependent block of putrescine, using the conductive state of the strong inwardly rectifying K+ channel GIRK2 as starting point. Our µs long simulations elucidate details about binding sites of putrescine in the pore and suggest that voltage-dependent rectification arises from a dual mechanism.

LUF7244 plus Dofetilide Rescues Aberrant Kv11.1 Trafficking and Produces Functional IKv11.1.

Voltage-gated potassium 11.1 (Kv11.1) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug-mediated Kv11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal Kv11.1 forward trafficking and thus reduce functional Kv11.1 channel densities. Although class III antiarrhythmics, e.g., dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use because of their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a Kv11.1 allosteric modulator/activator, to rescue Kv11.1 trafficking and produce functional Kv11.1 current. LUF7244 treatment by itself did not disturb or rescue wild type (WT) or G601S-Kv11.1 trafficking, as shown by Western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT Kv11.1 levels was rescued by 10 µM dofetilide or 10 µM dofetilide + 5 µM LUF7244. In trafficking defective G601S-Kv11.1 cells, dofetilide (10 µM) or dofetilide + LUF7244 (10 + 5 µM) also restored Kv11.1 trafficking, as demonstrated by Western blot and immunofluorescence microscopy. LUF7244 (10 µM) increased IKv 11.1 despite the presence of dofetilide (1 µM) in WT Kv11.1 cells. In G601S-expressing cells, long-term treatment (24-48 hour) with LUF7244 (10 µM) and dofetilide (1 µM) increased IKv11.1 compared with nontreated or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues Kv11.1 trafficking and produces functional IKv11.1 Thus, combined administration of LUF7244 and an IKv11.1 trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced Kv11.1 trafficking defects. SIGNIFICANCE STATEMENT: Decreased levels of functional Kv11.1 potassium channel at the plasma membrane of cardiomyocytes prolongs action potential repolarization, which associates with cardiac arrhythmia. Defective forward trafficking of Kv11.1 channel protein is an important factor in acquired and congenital long QT syndrome. LUF7244 as a negative allosteric modulator/activator in combination with dofetilide corrected both congenital and acquired Kv11.1 trafficking defects, resulting in functional Kv11.1 current.

Toward a Structural View of hERG Activation by the Small-Molecule Activator ICA-105574.

Outward current conducted by human ether-à-go-go-related gene type 1 (hERG1) K+ channels is important for action potential repolarization in the human ventricle. Rapid, voltage-dependent inactivation greatly reduces outward currents conducted by hERG1 channels and involves conformational changes in the ion selectivity filter (SF). Recently, compounds have been found that activate hERG1 channel function by modulating gating mechanisms such as reducing inactivation. Such activating compounds could represent a novel approach to prevent arrhythmias associated with prolonged ventricular repolarization associated with inherited or acquired long QT syndrome. ICA-105574 (ICA), a 3-nitro-n-(4-phenoxyphenyl) benzamide derivative activates hERG1 by strongly attenuating pore-type inactivation. We previously mapped the putative binding site for ICA to a hydrophobic pocket located between two adjacent subunits. Here, we used the recently reported cryoelectron microscopy structures of hERG1 to elucidate the structural mechanisms by which ICA influences the stability of the SF. By combining molecular dynamics simulations, voltage-clamp electrophysiology, and the synthesis of novel ICA derivatives, we provide atomistic insights into SF dynamics and propose a structural link between the SF and S6 segments. Further, our study highlights the importance of the nitro moiety, at the meta position of the benzamide ring, for the activity of ICA and reveals that the (bio)isosteric substitution of this side chain can switch the activity to weak inhibitors. Our findings indicate that ICA increases the stability of the SF to attenuate channel inactivation, and this action requires a fine-tuned compound geometry.

Atomistic basis of opening and conduction in mammalian inward rectifier potassium (Kir2.2) channels.

Potassium ion conduction through open potassium channels is essential to control of membrane potentials in all cells. To elucidate the open conformation and hence the mechanism of K+ ion conduction in the classic inward rectifier Kir2.2, we introduced a negative charge (G178D) at the crossing point of the inner helix bundle, the location of ligand-dependent gating. This "forced open" mutation generated channels that were active even in the complete absence of phosphatidylinositol-4,5-bisphosphate (PIP2), an otherwise essential ligand for Kir channel opening. Crystal structures were obtained at a resolution of 3.6 Å without PIP2 bound, or 2.8 Å in complex with PIP2. The latter revealed a slight widening at the helix bundle crossing (HBC) through backbone movement. MD simulations showed that subsequent spontaneous wetting of the pore through the HBC gate region allowed K+ ion movement across the HBC and conduction through the channel. Further simulations reveal atomistic details of the opening process and highlight the role of pore-lining acidic residues in K+ conduction through Kir2 channels.

Disease Associated Mutations in KIR Proteins Linked to Aberrant Inward Rectifier Channel Trafficking.

The ubiquitously expressed family of inward rectifier potassium (KIR) channels, encoded by KCNJ genes, is primarily involved in cell excitability and potassium homeostasis. Channel mutations associate with a variety of severe human diseases and syndromes, affecting many organ systems including the central and peripheral neural system, heart, kidney, pancreas, and skeletal muscle. A number of mutations associate with altered ion channel expression at the plasma membrane, which might result from defective channel trafficking. Trafficking involves cellular processes that transport ion channels to and from their place of function. By alignment of all KIR channels, and depicting the trafficking associated mutations, three mutational hotspots were identified. One localized in the transmembrane-domain 1 and immediately adjacent sequences, one was found in the G-loop and Golgi-export domain, and the third one was detected at the immunoglobulin-like domain. Surprisingly, only few mutations were observed in experimentally determined Endoplasmic Reticulum (ER)exit-, export-, or ER-retention motifs. Structural mapping of the trafficking defect causing mutations provided a 3D framework, which indicates that trafficking deficient mutations form clusters. These "mutation clusters" affect trafficking by different mechanisms, including protein stability.

Conduction through a narrow inward-rectifier K+ channel pore.

Inwardly rectifying potassium (Kir) channels play a key role in controlling membrane potentials in excitable and unexcitable cells, thereby regulating a plethora of physiological processes. G-protein-gated Kir channels control heart rate and neuronal excitability via small hyperpolarizing outward K+ currents near the resting membrane potential. Despite recent breakthroughs in x-ray crystallography and cryo-EM, the gating and conduction mechanisms of these channels are poorly understood. MD simulations have provided unprecedented details concerning the gating and conduction mechanisms of voltage-gated K+ and Na+ channels. Here, we use multi-microsecond-timescale MD simulations based on the crystal structures of GIRK2 (Kir3.2) bound to phosphatidylinositol-4,5-bisphosphate to provide detailed insights into the channel's gating dynamics, including insights into the behavior of the G-loop gate. The simulations also elucidate the elementary steps that underlie the movement of K+ ions through an inward-rectifier K+ channel under an applied electric field. Our simulations suggest that K+ permeation might occur via direct knock-on, similar to the mechanism recently shown for Kv channels.

LUF7244, an allosteric modulator/activator of Kv 11.1 channels, counteracts dofetilide-induced torsades de pointes arrhythmia in the chronic atrioventricular block dog model.

BACKGROUND AND PURPOSE: Kv 11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of Kv 11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model. EXPERIMENTAL APPROACH: LUF7244 was tested in vitro for (a) increasing human IKv11.1 and canine IKr and (b) decreasing dofetilide-induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block. KEY RESULTS: LUF7244 (0.5-10 µM) concentration dependently increased IKv11.1 by inhibiting inactivation. In vitro, LUF7244 (10 µM) had no effects on IKIR2.1 , INav1.5 , ICa-L , and IKs , doubled IKr , shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide-induced early afterdepolarizations. LUF7244 (2.5 mg·kg-1 ·15 min-1 ) in dogs with sinus rhythm was not proarrhythmic and shortened, non-significantly, repolarization parameters (QTc: -6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide-induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 µM after chronic atrioventricular block. CONCLUSIONS AND IMPLICATIONS: LUF7244 counteracted dofetilide-induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of Kv 11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening.

Computational Identification of Novel Kir6 Channel Inhibitors.

KATP channels consist of four Kir6.x pore-forming subunits and four regulatory sulfonylurea receptor (SUR) subunits. These channels couple the metabolic state of the cell to membrane excitability and play a key role in physiological processes such as insulin secretion in the pancreas, protection of cardiac muscle during ischemia and hypoxic vasodilation of arterial smooth muscle cells. Abnormal channel function resulting from inherited gain or loss-of-function mutations in either the Kir6.x and/or SUR subunits are associated with severe diseases such as neonatal diabetes, congenital hyperinsulinism, or Cantú syndrome (CS). CS is an ultra-rare genetic autosomal dominant disorder, caused by dominant gain-of-function mutations in SUR2A or Kir6.1 subunits. No specific pharmacotherapeutic treatment options are currently available for CS. Kir6 specific inhibitors could be beneficial for the development of novel drug therapies for CS, particular for mutations, which lack high affinity for sulfonylurea inhibitor glibenclamide. By applying a combination of computational methods including atomistic MD simulations, free energy calculations and pharmacophore modeling, we identified several novel Kir6.1 inhibitors, which might be possible candidates for drug repurposing. The in silico predictions were confirmed using inside/out patch-clamp analysis. Importantly, Cantú mutation C166S in Kir6.2 (equivalent to C176S in Kir6.1) and S1020P in SUR2A, retained high affinity toward the novel inhibitors. Summarizing, the inhibitors identified in this study might provide a starting point toward developing novel therapies for Cantú disease.

A structural model of the human serotonin transporter in an outward-occluded state.

The human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin. Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT.

Glibenclamide and HMR1098 normalize Cantú syndrome-associated gain-of-function currents.

Cantú syndrome (CS) is caused by dominant gain-of-function mutation in ATP-dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but IKATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that IKATP inhibitors, glibenclamide and HMR1098, normalize CS channels. IKATP in response to Mg-ATP, glibenclamide and HMR1098 were measured by inside-out patch-clamp electrophysiology. Results were interpreted in view of cryo-EM IKATP channel structures. Mg-ATP IC50 values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild-type (0.15 ± 0.01). HMR1098 dose-dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg-ATP, reaching, at 30 µmol/L, current levels displayed by wild-type and H60Y channels in the presence of 0.15 mmol/L Mg-ATP. Glibenclamide (10 µmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg-ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only.

Histidine at position 462 determines the low quinine sensitivity of ether-à-go-go channel superfamily member Kv 12.1.

BACKGROUND AND PURPOSE: The ether-à-go-go (Eag) Kv superfamily comprises closely related Kv 10, Kv 11, and Kv 12 subunits. Kv 11.1 (termed hERG in humans) gained much attention, as drug-induced inhibition of these channels is a frequent cause of sudden death in humans. The exclusive drug sensitivity of Kv 11.1 can be explained by central drug-binding pockets that are absent in most other channels. Currently, it is unknown whether Kv 12 channels are equipped with an analogous drug-binding pocket and whether drug-binding properties are conserved in all Eag superfamily members. EXPERIMENTAL APPROACH: We analysed sensitivity of recombinant Kv 12.1 channels to quinine, a substituted quinoline that blocks Kv 10.1 and Kv 11.1 at low micromolar concentrations. KEY RESULTS: Quinine inhibited Kv 12.1, but its affinity was 10-fold lower than for Kv 11.1. Contrary to Kv 11.1, quinine inhibited Kv 12.1 in a largely voltage-independent manner and induced channel opening at more depolarised potentials. Low sensitivity of Kv 12.1 and characteristics of quinine-dependent inhibition were determined by histidine 462, as site-directed mutagenesis of this residue into the homologous tyrosine of Kv 11.1 conferred Kv 11.1-like quinine block to Kv 12.1(H462Y). Molecular modelling demonstrated that the low affinity of Kv 12.1 was determined by only weak interactions of residues in the central cavity with quinine. In contrast, more favourable interactions can explain the higher quinine sensitivity of Kv 12.1(H462Y) and Kv 11.1 channels. CONCLUSIONS AND IMPLICATIONS: The quinoline-binding "motif" is not conserved within the Eag superfamily, although the overall architecture of these channels is apparently similar. Our findings highlight functional and pharmacological diversity in this group of evolutionary-conserved channels.