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BACKGROUND AND PURPOSE The ether-a-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.

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.

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+ flux. 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 Gβγ, 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.

Kv11.1 (hERG) 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 due to 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 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) restored Kv11.1 trafficking also, as demonstrated by western blot and immunofluorescence microscopy. LUF7244 (10 μM) increased IKv11.1 despite the presence of dofetilide (1 μM) in WT Kv11.1 cells. In G601S expressing cells, long-term treatment (24-48 h) with LUF7244 (10 μM) and dofetilide (1 μM) increased IKv11.1 compared to non-treated, 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.

Outward current conducted by human ether-à-go-go–related gene type 1 (hERG1) channels is a major determinant of action potential repolarization in the human ventricle. Ginsenoside 20(S)-Rg3 [Rg3; (2S,3R,4S,5S,6R)-2-[(2R,3R,4S,5S,6R)-4,5-dihydroxy-2-[[(3S,5R,8R,9R,10R,12R,13R,14R,17S)-12-hydroxy-17-[(2S)-2-hydroxy-6-methylhept-5-en-2-yl]-4,4,8,10,14-pentamethyl-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]oxy]-6-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol], an alkaloid isolated from the root of Panax ginseng, slows the rate of hERG1 deactivation, induces channels to open at more negative potentials than normal, and increases current magnitude. The onset of Rg3 action is extremely fast, suggesting that it binds to an extracellular accessible site on the channel to alter its gating. Here we used a scanning mutagenesis approach to identify residues in the extracellular loops and transmembrane segments of hERG1 that might interact with Rg3. Single or multiple residues of hERG1 were mutated to Ala or Cys and the resulting mutant channels were heterologously expressed in Xenopus oocytes. The effects of Rg3 on the voltage dependence of activation and the deactivation rate of mutant channel currents were characterized using the two-microelectrode voltage clamp technique. Mutation to Ala of specific residues in the S1 (Tyr420), S2 (Leu452, Phe463), and S4 (Ile521, Lys525) segments partially inhibited the effects of Rg3 on hERG1. The double mutant Y420A/L452A nearly eliminated the effects of Rg3 on voltage-dependent channel gating but did not prevent the increase in current magnitude. These findings together with molecular modeling suggest that Rg3 alters the gating of hERG1 channels by interacting with and stabilizing the voltage sensor domain in an activated state.

Outward current conducted by human ether-a-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.

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.

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 Cantú syndrome. Kir6 specific inhibitors could be beneficial for the development of novel drug therapies for Cantú syndrome, 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 modelling, 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 C176S in Kir6.1 and S1020P in SUR2A, retained high affinity towards the novel inhibitors. Summarizing, the inhibitors identified in this study might provide a starting point towards developing novel therapies for Cantú disease.

This paper presents the crystal structure of a forced open inward rectifier Kir2.2 channel. Molecular dynamics reveals the details of ion permeation through the open channel., 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.

EAG1 channels belong to the KCNH family of voltage gated potassium channels. They are expressed in several brain regions and increased expression is linked to certain cancer types. Recent cryo-EM structure determination finally revealed the structure of these channels in atomic detail, allowing computational investigations. In this study, we performed molecular dynamics simulations to investigate the ion binding sites and the dynamical behavior of the selectivity filter. Our simulations suggest that sites S2 and S4 form stable ion binding sites, while ions placed at sites S1 and S3 rapidly switched to sites S2 and S4. Further, ions tended to dissociate away from S0 within less than 20 ns, due to increased filter flexibility. This was followed by water influx from the extracellular side, leading to a widening of the filter in this region, and likely non-conductive filter configurations. Simulations with the inactivation-enhancing mutant Y464A or Na+ ions lead to trapped water molecules behind the SF, suggesting that these simulations captured early conformational changes linked to C-type inactivation.

Background/Aims: Lubeluzole is a benzothiazole derivative that has shown neuroprotective properties in preclinical models of ischemic stroke. However, clinical research on lubeluzole is now at a standstill, since lubeluzole seems to be associated with the acquired long QT syndrome and ventricular arrhythmias. Since the cardiac cellular effects of lubeluzole have not been described thus far, an explanation for the lubeluzole-induced QT interval prolongation is lacking. Methods: We tested the affinity of lubeluzole, its enantiomer, and the racemate for hERG channel using the patch-clamp technique. We synthesized and tested two simplified model compounds corresponding to two moieties included in the lubeluzole structure. The obtained experimental results were rationalized by docking simulation on the recently reported cryo-electron microscopy (cryo-EM) structure of hERG. Group efficiency analysis was performed in order to individuate the fragment most contributing to binding. Results: We found that lubeluzole and its R enantiomer are highly potent inhibitors of human ether-ago-go-related gene (hERG) channel with an IC50 value of 12.9 ± 0.7 nM and 11.3 ± 0.8 nM, respectively. In the presence of lubeluzole, steady-state activation and inactivation of hERG channel were shifted to more negative potentials and inactivation kinetics was accelerated. Mutations of aromatic residues (Y652A and F656A) in the channel inner cavity significantly reduced the inhibitory effect of lubeluzole. Molecular docking simulations performed on the near atomic resolution cryo-electron microscopy structures of hERG supported the role of Y652 and F656 as the main contributors to high affinity binding. Group efficiency analysis indicated that both 1,3-benzothiazol-2-amine and 3-aryloxy-2-propanolamine moieties contribute to drug binding with the former giving higher contribution. Conclusions: This study suggests the possibility to modulate lubeluzole hERG blockade by introducing suitable substituents onto one or both constituting portions of the parent compound in order to either reduce potency (i. e. torsadogenic potential) or potentiate affinity (useful for class III antiarrhythmic and anticancer agent development).

Background and Purpose Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied. Experimental Approach hERG and KIR2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr. Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues. Key Results Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and KIR2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not KIR2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective. Conclusions and Implications Drug-induced trafficking defects can be minimized if certain chemical features are avoided or ‘synthesized out’; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS.

Aims In excitable cells, KIR2.x ion-channel-carried inward rectifier current ( I K1) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific I K1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of I K1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits I K1 by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific I K1 inhibitors based on the P structure. Methods and results We analysed seven pentamidine analogues (PA-1 to PA-7) for I K1 blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC50 values (12–15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (Δ G −44.1 kJ/Mol) than for P (Δ G −31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect I Nav1.5, I Ca-L, I Kv4.3, I Kv11.1, and I Kv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of I K1 in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 ( n = 9), 26 ( n = 5), and 34% ( n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM. Conclusion PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete I K1 block.

Phospholipids are required to bind to two distinct sites on the inward rectifier potassium channel for maximal efficacy. Lee et al. show that a membrane-associating tryptophan residue in the second site can mimic the effect of phospholipid binding and cause a conformational change to reveal the primary binding site., Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL−) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL−. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL− binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL− binding and PIP2 sensitivity.

The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causing serious cardiac side effects, is a major problem in drug development. Despite the large amounts of existing biochemical data on blockade of hERG by drugs and druglike compounds, relatively little is known regarding the structural basis of binding of blockers to the channel. Here, we have used a recently developed homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations yield a remarkably good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships in terms of a number of key interactions. Two main interaction regions of the channel are thus identified with implications for further mutagenesis experiments and design of new compounds.

Single point mutations in pore-forming S6 segments of calcium channels may transform a high-voltage-activated into a low-voltage-activated channel, and resulting disturbances in calcium entry may cause channelopathies (Hemara-Wahanui et al., Proc Natl Acad Sci U S A 102(21):7553–7558, 16). Here we ask the question how physicochemical properties of amino acid residues in gating-sensitive positions on S6 segments determine the threshold of channel activation of CaV1.2. Leucine in segment IS6 (L434) and a newly identified activation determinant in segment IIIS6 (G1193) were mutated to a variety of amino acids. The induced leftward shifts of the activation curves and decelerated current activation and deactivation suggest a destabilization of the closed and a stabilisation of the open channel state by most mutations. A selection of 17 physicochemical parameters (descriptors) was calculated for these residues and examined for correlation with the shifts of the midpoints of the activation curve (ΔVact). ΔVact correlated with local side-chain flexibility in position L434 (IS6), with the polar accessible surface area of the side chain in position G1193 (IIIS6) and with hydrophobicity in position I781 (IIS6). Combined descriptor analysis for positions I781 and G1193 revealed that additional amino acid properties may contribute to conformational changes during the gating process. The identified physicochemical properties in the analysed gating-sensitive positions (accessible surface area, side-chain flexibility, and hydrophobicity) predict the shifts of the activation curves of CaV1.2. Electronic supplementary material The online version of this article (doi:10.1007/s00424-010-0885-2) contains supplementary material, which is available to authorized users.

The role of six cysteines of Ca(V)3.1 in channel gating was investigated. C241, C271, C282, C298, C313, and C323, located in the extracellular loop between segment IS5 and the pore helix, were each mutated to alanine; the resultant channels were expressed and studied by patch clamping in HEK293 cells. C298A and C313A conducted calcium currents, while the other mutants were not functional. C298A and C313A as well as double mutation C298/313A significantly reduced the amplitude of the calcium currents, shifted the activation curve in the depolarizing direction and slowed down channel inactivation. Redox agents dithiothreitol (DTT) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) shifted the current activation curve of wild-type channels in the hyperpolarizing direction. Activation curve for all mutated channels was shifted in hyperpolarizing direction by DTT while DTNB caused a depolarizing shift. Our study reveals that the cysteines we studied have an essential role in Ca(V)3.1 gating. We hypothesize that cysteines in the large extracellular loop of Ca(V)3.1 form bridges within the loop and/or neighboring channel segments that are essential for channel gating.

The hERG cavity can trap very bulky compounds, without perturbing normal gate closure., Inhibition of hERG K+ channels by structurally diverse drugs prolongs the ventricular action potential and increases the risk of torsade de pointes arrhythmias and sudden cardiac death. The capture of drugs behind closed channel gates, so-called drug trapping, is suggested to harbor an increased pro-arrhythmic risk. In this study, the trapping mechanisms of a trapped hERG blocker propafenone and a bulky derivative (MW: 647.24 g mol–1) were studied by making use of electrophysiological measurements in combination with molecular dynamics simulations. Our study suggests that the hERG cavity is able to accommodate very bulky compounds without disturbing gate closure.

K(+) channels play a critical role in numerous physiological and pathophysiological processes rendering them an attractive target for therapeutic intervention. However, the hERG K(+) channel poses a special challenge in drug discovery, since block of this channel by a plethora of diverse chemical entities can lead to long QT syndrome and sudden death. Of particular interest is the so-called trapping phenomenon, characterized by capture of a drug behind closed channel gates, which harbors an increased pro-arrhythmic risk. In this study we investigated the influence of trapped blockers on the gating dynamics and probed the state dependence of dissociation in K(+) channels by making use of the quaternary tetrabutylammonium. By applying essential dynamics simulations and two-electrode voltage clamp we obtained detailed insights into the dynamics of trapping in KcsA and hERG. Our simulations suggest that the trapped TBA influences the F656 flexibility during gate closure. Based on these findings, we provide a structural hypothesis for drug trapping. Further our simulations reveal the extent of gate opening necessary for drug dissociation.

Despite the availability of several crystal structures of bacterial voltage-gated Na(+) channels, the structure of eukaryotic Na(+) channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na(+) channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na(+) channels.

Understanding the molecular basis of hERG (Kv11.1) inhibition is essential for drug development. Here we systematically analyzed the mechanism and potency of hERG inhibition by a library of 13 dofetilide derivatives1. Currents through hERG channels stably expressed in HEK 293 cells were studied applying whole-cell configuration using planar patch clamp technique2.The estimated IC50s ranged between 4.3±0.7nM (LUF6200) and 297±77nM (LUF6139). An inverse relation was observed between potency (IC50) and rate of block development. Analysis of the kinetics of block development revealed that potency of dofetilide derivatives is predominantly determined by the drug dissociation rate constant. In general, higher molecular weight led to higher apparent drug affinity (r = 0.9).LUF6200, the most active compound, has 2 nitro groups in para position of the two aromatic rings and an ethyl moiety at the basic nitrogen atom. Pursuing a Topliss approach at the phenoxy group further supports the positive contribution of the nitro group1, showing that substituents with electron withdrawing groups in para position of the phenyl ring are beneficial for hERG blocking activity. This points towards a pi-pi interaction of at least one of the aromatic rings of dofetilide analogs with respective aromatic amino acids in the channel. Docking of the set of compounds into a homology model of the hERG channel supports this finding. A refined molecular model for dofetilide-hERG interaction based on the novel functional data and mutational studies is proposed.1. Shagufta et al. Exploring Chemical Substructures Essential for hERG K+ Channel Blockade by Synthesis and Biological Evaluation of Dofetilide Analogues. ChemMedChem 4, 1722-1732 (2009).2. Farre, C. & Fertig, N. HTS techniques for patch clamp-based ion channel screening - advances and economy. Expert Opin. Drug Discov. 7, 515-524 (2012).

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go-related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells. Currents through hERG channels were blocked regardless of whether ibogaine was applied via the extracellular or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during activation of the channels and was not observed for resting channels. With increasing depolarizations, ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, whereas inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, these findings show that ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.

The bacterial potassium channel KcsA, which has been crystallized in several conformations, offers an ideal model to investigate activation gating of ion channels. In this study, essential dynamics simulations are applied to obtain insights into the transition pathways and the energy profile of KcsA pore gating. In agreement with previous hypotheses, our simulations reveal a two phasic activation gating process. In the first phase, local structural rearrangements in TM2 are observed leading to an intermediate channel conformation, followed by large structural rearrangements leading to full opening of KcsA. Conformational changes of a highly conserved phenylalanine, F114, at the bundle crossing region are crucial for the transition from a closed to an intermediate state. 3.9 µs umbrella sampling calculations reveal that there are two well-defined energy barriers dividing closed, intermediate, and open channel states. In agreement with mutational studies, the closed state was found to be energetically more favorable compared to the open state. Further, the simulations provide new insights into the dynamical coupling effects of F103 between the activation gate and the selectivity filter. Investigations on individual subunits support cooperativity of subunits during activation gating., Author Summary Voltage gated ion channels are membrane embedded proteins that initiate electrical signaling upon changes in membrane potential. These channels are involved in biological key processes such as generation and propagation of nerve impulses. Mutations may lead to serious diseases such as cardiac arrhythmia, diabetes or migraines, rendering them important drug targets. The activity of ion channels is controlled by dynamic conformational changes that regulate ion flow through a central pore. This process, which involves opening and closing of the channels, is known as gating. To fully understand or to control ion channel gating, we need to unravel the underlying principles. Crystal structures, especially of K+ channels, have provided excellent insights into the conformation of different channel states. However, the transition states and structural rearrangements are still unknown. Here we use molecular dynamics simulations to simulate the full transition pathway and energy landscape of gating. Our results suggest that channel gating involves local structural changes followed by global conformational changes. The importance of many of the residues identified in our simulations is supported by experimental studies. The ability to accurately simulate the gating transitions of ion channels may be beneficial for a better understanding of ion channel related diseases and drug development.

Human ether-a-go-go related gene (hERG) channel inhibitors can be trapped in the channels at rest. The structural peculiarities of hERG blockers that enable trapping or alternatively resting state dissociation are currently unknown. Propafenone (small molecule, MW 341 g/mol) is efficiently trapped in the closed hERG channel pore (1). To investigate whether the size of the blocking molecule plays a role in trapping we synthesized bulky propafenone derivatives containing benzoyl and trimethylphenyl side chains, attached by piperazine linkers, with molecular weights of 500 (Fba212) and 650 g/mol (Fba213) respectively. hERG channels were expressed in Xenopus laevis oocyte and potassium current inhibition was studied using the two - microelectrode voltage clamp technique.It was found: first, both compounds are potent hERG blockers with IC50 3.7μM (Fba212) and 52μM (Fba213). Secondly, channel block by Fba212 and 213 was prevented by mutations Y652A and F656A as previously shown for propafenone. Third, both propafenone derivatives were trapped at rest. To obtain insights into the molecular mechanism of channel block docking experiments with Fba212 and Fba213 in closed and open conformation were performed. Both compounds interact with the propafenone binding site (Y652A and F656A). Fba213 was found to exceed the size of the closed channel cavity of our hERG homology model. We conclude that drug trapping in hERG channels does not necessarily require full closure of the activation gate.1. Witchel HJ, Dempsey CE, Sessions RB, Perry M, Milnes JT, Hancox JC, Mitcheson JS.Mol Pharmacol. 2004 Nov; 66(5):1201-12.

Rapid and selective ion transport is essential for the generation and regulation of electrical signalingpathways in living organisms. In this study, we use molecular dynamics simulations and free energy cal-culations to investigate how the bacterial sodium channel Na V Ab (Arcobacter butzleri) differentiatesbetween Na + and Ca 2+ ions. Multiple nanosecond molecular dynamics simulations revealed distinct bind-ing patterns for these two cations in the selectivity filter and suggested a high affinity calcium bindingsite formed by backbone atoms of residues Leu-176 and Thr-175 (S CEN ) in the sodium channel selectivityfilter. 2012 Elsevier Inc. All rights reserved. 1. IntroductionNa + flux is vital for initiating action potentials in the mem-branes of most electrically excitable cells [1]. Recent homotetra-meric crystal structures of bacterial Na V channels [2–5] providenew possibilities for investigating the molecular basis of ion selec-tivity and transport in these channels. The mechanisms of howthese channels discriminate between different ion types are poorlyunderstood. Recent molecular dynamics investigations [6,7] pro-vide insights into Na

Cardiac inward rectifier channels belong to three different classes of the K IR channel protein family. The K IR 2.x proteins generate the classical inward rectifier current, I K1 , while K IR 3 and KL IR 6 members are responsible for the acetylcholine responsive and ATP sensitive inward rectifier currents I KAch and I KATP , respectively. Aberrant function of these channels has been correlated with severe cardiac arrhythmias, indicating their significant contribution to normal cardiac electrophysiology. A common feature of inward rectifier channels is their dependence on the lipid phosphatidyl-4,5-bisphospate (PIP2) interaction for functional activity. Cationic amphiphilic drugs (CADs) are one of the largest classes of pharmaceutical compounds. Several widely used CADs have been associated with inward rectifier current disturbances, and recent evidence points to interference of the channel-PIP 2 interaction as the underlying mechanism of action. Here, we will review how six of these well known drugs, used for treatment in various different conditions, interfere in cardiac inward rectifier functioning. In contrast, K IR channel inhibition by the anionic anesthetic thiopental is achieved by a different mechanism of channel-PIP 2 interference. We will discuss the latest basic science insights of functional inward rectifier current characteristics, recently derived K IR channel structures and specific PIP 2 -receptor interactions at the molecular level and provide insight in how these drugs interfere in the structure-function relationships.

Voltage gated K+ channels open and close in response to voltage changes. While crystal structures capture a limited number of gating conformations (open, closed), the transition steps and mechanism are still unknown. A limited number of voltage gated potassium channels structures in either open or closed conformation have been crystallized. Since the bacterial K+ channel KcsA has been crystallized in open, intermediate (Cuello et al., 2010) and closed (Zhou et al., 2001) states, this channel offers an ideal model for pore gating simulations. Consequently, we applied essential dynamics simulations to study the gating pathway and energetics underlying pore gating. Essential dynamics simulations were performed at timescales ranging from 1 to 100 ns. Gating simulations were considered complete when the RMSD was below 1.5 A to the target structure. This criterion was reached in most runs, however shorter simulation times usually resulted in lower RMSD values. In agreement with structural data (Cuello et al., 2010), all available KcsA transition structures (PDB identifier: 3fb5, 3fb6, 3f7y) were sampled with our essential dynamics protocol. Furthermore, molecular dynamics runs revealed that channel opening and closing sampled the same conformations along the transition pathway. Removal of pH-Sensor residues (H25, E120, R121, R122, H124) led to spontaneous channel openings on the nanosecond timescale. These unbiased opening simulations sampled the same conformations as our essential dynamics simulations, further supporting the validity of our method. Finally, umbrella sampling was used to estimate the energy profiles of the various gating states and the transition pathways of WT and mutant channels. As expected, the energy needed to open the channel is significantly decreased in the pH-Sensor deleted KcsA.

Pharmacological inhibition of cardiac hERG K(+) channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652) substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences.

Recent crystal structures of the bacterial potassium channel KcsA have shed light on the intermediate and open conformation of the channel (Cuello et al. 2010). Although crystal structures provide excellent insights into different conformations of proteins, they feature only snapshots of dynamical proteins. Therefore, the transition steps and the mechanisms of activation gating are still unknown. In order to elucidate the activation gating dynamics KcsA, we applied essential dynamics (ED) simulations. The successful simulation of activation gating allowed us to calculate the underlying energy landscape by umbrella sampling. The energy profile revealed three energy wells that are separated by two barriers. We identified structural rearrangements of F114 which correspond the first energy barrier. The change in rotameric state of F114 is crucial for initial pore opening. Regarding the second energy barrier, we observed large conformational changes of the TM2 helices which represent the main final activation gate opening. Cooperativity of activation gating was studied by applying our ED protocol to different number of subunits (SUs). Our simulations showed that the movement of a single SU is not sufficient to open the activation gate. But by moving three SUs by ED simulations, the activation gate opened to the same extent as in the four SUs ED simulation protocol. These finding is in line with fluorescence detection studies, which showed that the SUs act cooperatively during gating (Blunck et al. 2008).

Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. Here, we use molecular dynamics (MD) simulations with an applied membrane potential to investigate the ion flux of bacterial sodium channel NaVMs. 5.9 µs simulations with 500 mM NaCl suggest different mechanisms for inward and outward flux. The predicted inward conductance rate of ∼27±3 pS, agrees with experiment. The estimated outward conductance rate is 15±3 pS, which is considerably lower. Comparing inward and outward flux, the mean ion dwell time in the selectivity filter (SF) is prolonged from 13.5±0.6 ns to 20.1±1.1 ns. Analysis of the Na+ distribution revealed distinct patterns for influx and efflux events. In 32.0±5.9% of the simulation time, the E53 side chains adopted a flipped conformation during outward conduction, whereas this conformational change was rarely observed (2.7±0.5%) during influx. Further, simulations with dihedral restraints revealed that influx is less affected by the E53 conformational flexibility. In contrast, during outward conduction, our simulations indicate that the flipped E53 conformation provides direct coordination for Na+. The free energy profile (potential of mean force calculations) indicates that this conformational change lowers the putative barriers between sites SCEN and SHFS during outward conduction. We hypothesize that during an action potential, the increased Na+ outward transition propensities at depolarizing potentials might increase the probability of E53 conformational changes in the SF. Subsequently, this might be a first step towards initiating slow inactivation., Author Summary Voltage gated sodium channels are essential components of living cell membranes. They regulate the cell potential by facilitating permeation of ions across the membrane. In the past decades, studies revealed that the bacterial selectivity filter (SF) exhibits a constricted architecture lined with electronegative carboxyl oxygens of four glutamic acid side chains (EEEE motif), which repulse anions but attract Na+ ions. Crystal structures enable the investigation of structural dynamics with computational methods. Ion selectivity and conduction mechanisms between Na+, K+ and Ca2+ are progressively elucidated by molecular dynamics simulations and free energy calculations. The structural dynamics of the protein, especially the flexibility of SF and its fundamental role in kinetics underpinning ion selectivity, conduction and channel gating are less well understood. To shed light on this question, we use computational simulations to simulate ion conduction with membrane potentials. Our results suggest different dynamical behaviors of the EEEE locus and distinct ion distribution patterns in the SF with respect to permeating directionalities. These findings indicate a novel mechanism in differentiating reciprocal transitions of ion flow, preventing large sodium efflux during action potential initiation and may further suggest that increased flipping propensities at depolarizing potentials, might initially trigger channel slow inactivation.

Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. Here, we use molecular dynamics (MD) simulations with applied membrane potential to investigate the ion flux of bacterial sodium channel NaVMs (Mccusker et al., 2012). 4-microsecond simulations with 500mM NaCl suggest different mechanisms for inward and outward flux.The predicted inward conductance rate of ∼26 pS agrees with experiment (∼33 pS, Ulmschneider et al., 2013). The estimated outward conductance rate is 14 pS, which is considerably slower. The mean ion dwell time of the selectivity filter is prolonged from 14.8±0.8 ns to 21.4±1.1 ns. Analysis of the Na+ distribution revealed distinct patterns for influx and efflux events.During influx, site-HFS (High Field Strength) (Payandeh et al., 2012) is the dominant site with highest ion density. Ions are directly coordinated by GLU53 and SER54 sidechains in an off-axis manner. Site-CEN and Site-IN are less populated. In contrast to site-HFS, ions are distributed in the middle of the selectivity filter.The distribution of the outward current displays a different pattern: site-CEN and site-IN are instantaneously occupied with Na+, while site HFS is populated less frequently.Examining the structural fluctuation of the trajectories suggests subtle differences at the selectivity filter. Translocation of ions from cytoplasma to periplasma induces structural changes of the GLU53 sidechains from an out-facing to a lumen-facing conformation. Thereby, it slows down the flux rate by creating a narrower constriction “mouth”. During influx this residue remains rigid and potentially energetically more favorable for ion passage. We hypothesize that these structural changes at the selectivity filter glutamic acids represent a novel gating mechanism decreasing outward current.

Voltage sensors initiate the closed - open transitions in the ion conducting pore. Here we consecutively neutralized the charges of the four S4 segments (IS4-IVS4) of CaV1.2. The channel construct lacking charges in IIS4 (IIS4N) conducted barium currents. Surprisingly, charge neutralization accelerated channel closure and diminished shifts of the activation curves also in S6 mutants (IS6: G432W/IIS4N, IIS6: A780T/IIS4N, IIIS6: G1193T/IIS4N, IVS6: A1503G/IIS4N). Such a rescue of S6-mediated gating perturbations is exemplified for the IVS6 mutation A1503G. A1503G induced a −22.6±1.2 mV shift of the activation curve to the left that was diminished by charge neutralization in IIS4. Gating disturbances induced by pore mutations upstream and downstream of G432, G1193 and A1503 were unaffected. Thermodynamic analysis reveals that segment IIS4 interacts (energetically coupled) with a ring of small homologous amino acids G432, A780, G1193 and A1503 (G/A/G/A ring, Depil et al. 2011) in all four pore forming S6 segments.The research was funded by the Austrian Science Fund (FWF): grant P22600-B12 and P19614.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Voltage sensors trigger the closed-open transitions in the pore of voltage-gated ion channels. To probe the transmission of voltage sensor signalling to the channel pore of Ca(V)1.2, we investigated how elimination of positive charges in the S4 segments (charged residues were replaced by neutral glutamine) modulates gating perturbations induced by mutations in pore-lining S6 segments. Neutralisation of all positively charged residues in IIS4 produced a functional channel (IIS4(N)), while replacement of the charged residues in IS4, IIIS4 and IVS4 segments resulted in nonfunctional channels. The IIS4(N) channel displayed activation kinetics similar to wild type. Mutations in a highly conserved structure motif on S6 segments ("GAGA ring": G432W in IS6, A780T in IIS6, G1193T in IIIS6 and A1503G in IVS6) induce strong left-shifted activation curves and decelerated channel deactivation kinetics. When IIS4(N) was combined with these mutations, the activation curves were shifted back towards wild type and current kinetics were accelerated. In contrast, 12 other mutations adjacent to the GAGA ring in IS6-IVS6, which also affect activation gating, were not rescued by IIS4(N). Thus, the rescue of gating distortions in segments IS6-IVS6 by IIS4(N) is highly position-specific. Thermodynamic cycle analysis supports the hypothesis that IIS4 is energetically coupled with the distantly located GAGA residues. We speculate that conformational changes caused by neutralisation of IIS4 are not restricted to domain II (IIS6) but are transmitted to gating structures in domains I, III and IV via the GAGA ring.

The chloride channel (CLC) family is distinctive in that some members are Cl(-) ion channels and others are Cl(-)/H(+) antiporters. The molecular mechanism that couples H(+) and Cl(-) transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl(-) and H(+) transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl(-)-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl(-) transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO(4)(2-) strongly competes with Cl(-) uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl(-)/H(+) antiporter, even though it contains an isoleucine at the Glu(in) position that was previously thought to be a critical part of the H(+) pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Glu(in) site in CLC-ck2 does not increase H(+) flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu(ex)) in CLC-ck2 prevents H(+) flux. Hence, Glu(ex), but not Glu(in), is critical for H(+) permeation in CLC proteins.

ICA105574 (3-Nitro-N-(4-phenoxyphenyl)-benzamide) activates hERG1 (Kv11.1) channels by reducing inactivation, but inhibits hEAG1 (Kv10.1) channels by inducing inactivation. using chimeric ERG-EAG constructs, we found that the channel-specific response to ICA105574 is mediated by the pore region (S5-pore helix-S6). Simulated docking of ICA105574 on a molecular model of hEAG1 coupled with alanine mutagenesis indicates that similar to hERG1, ICA105574 binds to a hydrophobic region confined by the S5/S6/pore helix of one subunit and S6 of adjacent subunit in hEAG1 channels. Based on the putative ICA binding site and protein sequence alignment, a triple mutant of hEAG1 (M431F/M458L/L463M hEAG1) was designed to make the ICA binding region in hEAG1 homologous to hERG1. ICA inhibited the triple mutant similar to WT-hEAG1, indicating that the amino acid differences within the putative ICA binding region do not determine the reciprocal functional response to ICA. A single mutation in S5 (F557L), pore helix (L622C) or S6 (Y652A) of hERG1 eliminates the activity of ICA on hERG1 (Garg, et al 2011, Mol. Pharm.). The equivalent mutations in hEAG1 affect inactivation differentially and result in very different responses to ICA. First, Y464A induced inactivation of hEAG1 channels and this gating was accentuated by ICA. Second, ICA is a partial agonist of F359L and L434C mutant channels. We recently proposed a tripartite model of slow inactivation for hEAG1 that involves interaction of the same three residues. We conclude that ICA binds to the same site in hEAG1 and hERG1 channels, and that the functional consequences are determined by the underlying (fast or slow) mode of selectivity-filter mediated inactivation.

Voltage-gated Na+ (NaV) are required for rapid signal propagation in cardiac, muscle and nerve cells. How NaV channels distinguish between sodium and calcium is an unresolved question. Selectivity is assumed to involve subtle mechanisms, since both ions have nearly identical size.Interestingly, the selectivity filter of bacterial NaV channels is expected to be quite similar with the filter of voltage gated calcium channels, since both contain a highly conserved selectivity filter signature sequence (TxExW). Despite this, bacterial NaV channels are selective for sodium with permeability ratios PCa/PNa of ∼0.08-0.15 (Shaya et al. 2011).To investigate how bacterial NaVAb channels (Payandeh et al. 2011) discriminate between Ca2+ and Na+, we performed molecular dynamics simulations.Firstly, double bilayer simulations with varying charge gradients and 200mM NaCl concentration were performed. As expected, rapid Na+ influx is observed. Simulations with 200mM CaCl2 revealed that Ca2+ ions can move into the selectivity filter, but not into the cavity, consequently no ion flux was observed. Additionally, calcium ions induced conformational changes of the glutamate side chain, which is in contrast to NaCl simulations, where the filter remained unchanged. To get detailed insights into this surprising behavior, we performed single-ion potential of mean force (PMF) calculations with Na+ and Ca2+.PMF simulations revealed that there is no energy barrier higher than 2.1 kcal/mol for a Na+ ion, suggesting that it can easily pass the filter. In contrast, Ca2+ encounters an energy barrier of approximately 13.8 kcal/mol, preventing calcium ions from moving into the cavity.Results of our simulations are consistent with a recent X-ray structure on the NaVRh channel (Zhang et al. 2012), showing a Ca2+ ion bound to the same site of the selectivity filter as seen in this study for NaVAb.

Results Homology modeling revealed that G432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (G432 (IS6), A780 (IIS6), G1193 (IIIS6), A1503 (IVS6)). In contrast, corresponding mutations in domains II, III and IV induced parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of G432 in domain I. Mutations of G432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices.