Affinity For Channels Research Articles

Scorpion venom increases acetylcholine release by prolonging the duration of somatic nerve action potentials

Scorpionism is frequently accompanied by a massive release of catecholamines and acetylcholine from peripheral nerves caused by neurotoxic peptides present in these venoms, which have high specificity and affinity for ion channels. Tityus bahiensis is the second most medically important scorpion species in Brazil but, despite this, its venom remains scarcely studied, especially with regard to its pharmacology on the peripheral (somatic and autonomic) nervous system. Here, we evaluated the activity of T. bahiensis venom on somatic neurotransmission using myographic (chick and mouse neuromuscular preparations), electrophysiological (MEPP, EPP, resting membrane potentials, perineural waveforms, compound action potentials) and calcium imaging (on DRG neurons and muscle fibres) techniques. Our results show that the major toxic effects of T. bahiensis venom on neuromuscular function are presynaptically driven by the increase in evoked and spontaneous neurotransmitter release. Low venom concentrations prolong the axonal action potential, leading to a longer depolarization of the nerve terminals that enhances neurotransmitter release and facilitates nerve-evoked muscle contraction. The venom also stimulates the spontaneous release of neurotransmitters, probably through partial neuronal depolarization that allows calcium influx. Higher venom concentrations block the generation of action potentials and resulting muscle twitches. These effects of the venom were reversed by low concentrations of TTX, indicating voltage-gated sodium channels as the primary target of the venom toxins. These results suggest that the major neuromuscular toxicity of T. bahiensis venom is probably mediated mainly by α- and β-toxins interacting with presynaptic TTX-sensitive ion channels on both axons and nerve terminals.

A A Review – Rasasindur Various Types of Anupanas w.r.t. Rastangini

Ras Sindoor is an Ayurvedic medicine, with herbal and mineral ingredients, in powder form. Anupana is explained by all Bruhatrayis in detail.Those substances which are drunk along with or after medicine or food intake is known as anupana. Ayurveda also teaches us that certain substances have a particular affinity for specific tissue and channels in the body. A carrier substance (anupan or anupana, in Sanskrit) can be used to help catalyze the delivery of a particular herb or formula to a specific tissue or channel, to encourage its penetration deep into the cells, or to moderate its effect in some way. In this paper introduce Rassindur and various types of anupan use in various diseases.

Real-time gesture recognition based on feature recalibration network with multi-scale information

Gesture recognition is important in human–machine interaction. Current methods for solving gesture recognition have several disadvantages such as low recognition rate, slow speed and poor performance on recognizing multiple targets or long-distance targets in complex environments. In view of the above problems, we propose a gesture recognition approach that can recognize gestures quickly and accurately from complex background. This approach works on a deep convolutional network, which consists of a basic network module for extracting feature information, a squeeze-and-excitation networks for increasing feature channel affinity and a feature pyramid attention module for fusing context information with different scales. To test the proposed approach, we make a data set that contains 3289 images from difference complex scenes. Generally gestures in those images can be generally classified into 16 types. We have uploaded this data set for researchers use. Experimental results demonstrate that the recognition accuracy and speed of the proposed method can achieve 83.45% and 32.2 frames per second respectively, which has better comprehensive performance compared with other state-of-the-art recognition algorithms.

IP3 R1-Mediated Local Ca2+ Release Events are Enhanced in the Gain-of-Function D2594K Mutant Channel

Quantal Ca2+ release through the inositol 1,4,5-trisphosphate receptor (IP3R) is a well-defined and ubiquitous phenomenon. However, its molecular origin remains poorly understood. Our general assumption is that this process results from variations of the channel's IP3 affinity as a function of the intra-pore [Mg2+]/[Ca2+] ratio. This ratio may vary during Ca2+ release and could be sensed by a residue (D2594) located in the inner portion of the channel's permeation pore. The hypothesis tested in this work is that the gain-of-function behavior shown by the IP3R when the D2594 residue is replaced by the basic residue K, is explained by increased IP3 affinity of the channel. If so, we predicted this mutation would modify the biophysical attributes of the resulting local Ca2+ release events (puffs) resulting in larger, brighter, and/or longer puffs. To test this, Ca2+ puffs were recorded with real-time, fast Ca2+-imaging, confocal microscopy after perfusing the purinergic ligand ATP (30 & 50 nM) and puff biophysical attributes analyzed. Experiments were conducted in intact HEK cells expressing either WT or D2594K mutant IP3R. Our results indicate that D2594K transfected intact HEK cells exhibited Ca2+ puffs with no significant differences in duration, but more than 6-fold larger amplitude (WT 0.88 ± 0.24 deltaF/F0; D2594 6.1 ± 0.24 deltaF/F0) than HEK cells expressing the WT channel. In addition, D2594K-expressing cells showed more than 1.5-fold higher Ca2+ puff frequency at every tested [ATP] (WT: 2.8 & 3.3 puffs/cell⋅min at 30 & 50 nM ATP, respectively. D2594: 4.8 & 5.3 puffs/cell⋅min at 30 & 50 nM ATP, respectively). These results are consistent with our hypothesis that quantal IP3 R-mediated Ca2+ release is controlled by the D2594 residue which allosterically affects the channel's affinity for IP3.

Removal of hERG potassium channel affinity through introduction of an oxygen atom: Molecular insights from structure-activity relationships of strychnine and its analogs

Nux vomica has been effectively used in Traditional Chinese Medicine. The processing of Nux vomica is necessary to reduce toxicity before it can be used in clinical practice. However, the mechanism for processing detoxification is unclear. hERG channels have been subjected to a routine test for compound cardiac toxicity in the drug development process. Therefore, we examined the effects and mechanisms of strychnine and brucine, two main ingredients of Nux vomica, and their N-oxides on hERG channels. Strychnine and brucine exhibited concentration-dependent inhibition of hERG channels with IC50 values of 25.9 μM and 44.18 μM, respectively. However, their nitrogen oxidative derivatives produced by processing of Nux vomica, strychnine N-oxide and brucine N-oxide, lost their activity on hERG channels. Compared to their parent compounds, only an oxygen atom was introduced in the nitrogen oxidative isoforms to compensate for the N+ − charge, suggesting that the protonated nitrogen is the key group for strychnine and brucine binding to hERG channel. Alanine-mutagenesis identified Y652 is the most important residue for strychnine and brucine binding to hERG channel. Y652A mutation increased the IC50 for strychnine and brucine by 21.64-fold and 29.78-fold that of WT IhERG, respectively. Docking simulations suggested that the protonated nitrogen of strychnine and brucine formed a cation–π interaction with the aromatic ring of Y652. This study suggests that introduction of an oxygen to compensate for the N+ − charge could be a useful strategy for reducing hERG potency and increasing the safety margin of alkaloid-type compounds in drug development.

Evaluation of anticonvulsant and analgesic activity of new hybrid compounds derived from N-phenyl-2-(2,5-dioxopyrrolidin-1-yl)-propanamides and –butanamides

Epilepsy is a chronic neurological disorder that is associated with various types of recurrent seizures, which are drug-resistant in about one third of patients. Moreover, anticonvulsant drugs are used to treat a wide range of non-epileptic conditions, including chronic pain. Here, we investigated the anticonvulsant activity of six new hybrid compounds based on the pyrrolidine-2,5-dione scaffold in the 6 Hz corneal stimulation test with 44 mA stimulus intensity in mice, which is the model of pharmacoresistant seizures. We demonstrated that two molecules, DK-10 (11) and DK-14 (14) show higher anticonvulsant activity and similar safety profile in comparison with valproic acid and much higher in comparison with levetiracetam in the aforementioned test. The second aim of this study was to examine analgesic activity of these compounds. For this purpose, the hot plate test, the formalin test, and the oxaliplatin-induced peripheral neuropathy model were performed. Among tested agents DK-11 (12) revealed prominent antinociceptive activity at non-sedative doses in the second (inflammatory) phase of the formalin test, which is the model of tonic pain and antiallodynic activity in the oxaliplatin-induced neuropathic pain, the model of painful chemotherapy-induced peripheral neuropathy. No cytotoxic effect on hepatoma cells was observed. Compound DK-10 (11) had high affinity for voltage-gated sodium channels, whereas compound DK-11 (12) showed weak binding toward sodium and calcium voltage-gated channels and the NMDA receptor. As a result, hybrid compounds reported herein seem to be very promising broad spectrum anticonvulsant molecules with collateral analgesic activity.

The effects of barnidipine on an experimental ischemia reperfusion model of spinal cord injury and comparison with methyl prednisolone.

OBJECTIVE:Increased intracellular calcium concentration plays an important role in the secondary mechanism of spinal cord injury. In the presenting experimental study, we aimed to evaluate the healing effect of barnidipine, which has a high affinity for L-type calcium channels, on acute spinal cord injury and to compare its effects with those of methylprednisolone.METHODS:A total of 32 Spraque Dawley albino adult female rats were divided into 4 groups; group 1: sham-operated (n=8), group 2: only ischemia (n=6), group 3: barnidipine-treated (n=8), and group 4: methylprednisolone-treated (n=6). An ischemia-reperfusion model was created by clipping the abdominal aorta in the rats. Motor examination was performed 1 hour after the surgical procedure and before sacrification. Immediately following the second motor examination, rats were sacrificed and tissue samples were taken for histopathological examination and for testing of tissue malondialdehyde (MDA) levels.RESULTS:A significant correlation of motor examination was found between the sham-operated and barnidipine-treated groups and the sham-operated and only ischemia groups at the 1st and 24th hour (p<0.008). There was no significant difference between the only ischemia and barnidipine-treated groups and only ischemia and methylprednisolone-treated groups (p>0.008). Light microscopic examination of the sham-operated group revealed findings consistent with normal spinal cord structure. In group 2, 3, and 4, light microscopic examination revealed polymorphonuclear leukocyte infiltration and a small amount of axonal swelling. There was no significant correlation between the ischemia and barnidipine-treated groups and the barnidipine and methylprednisolone groups in terms of MDA levels (p>0.008).CONCLUSION:A single dose of barnidipine (10 mg/kg) and methylprednisolone are not effective and not sufficient to prevent spinal ischemia-reperfusion injury in rats.

Guanidinium Toxins and Their Interactions with Voltage-Gated Sodium Ion Channels.

Guanidinium toxins, such as saxitoxin (STX), tetrodotoxin (TTX) and their analogs, are naturally occurring alkaloids with divergent evolutionary origins and biogeographical distribution, but which share the common chemical feature of guanidinium moieties. These guanidinium groups confer high biological activity with high affinity and ion flux blockage capacity for voltage-gated sodium channels (NaV). Members of the STX group, known collectively as paralytic shellfish toxins (PSTs), are produced among three genera of marine dinoflagellates and about a dozen genera of primarily freshwater or brackish water cyanobacteria. In contrast, toxins of the TTX group occur mainly in macrozoa, particularly among puffer fish, several species of marine invertebrates and a few terrestrial amphibians. In the case of TTX and analogs, most evidence suggests that symbiotic bacteria are the origin of the toxins, although endogenous biosynthesis independent from bacteria has not been excluded. The evolutionary origin of the biosynthetic genes for STX and analogs in dinoflagellates and cyanobacteria remains elusive. These highly potent molecules have been the subject of intensive research since the latter half of the past century; first to study the mode of action of their toxigenicity, and later as tools to characterize the role and structure of NaV channels, and finally as therapeutics. Their pharmacological activities have provided encouragement for their use as therapeutants for ion channel-related pathologies, such as pain control. The functional role in aquatic and terrestrial ecosystems for both groups of toxins is unproven, although plausible mechanisms of ion channel regulation and chemical defense are often invoked. Molecular approaches and the development of improved detection methods will yield deeper understanding of their physiological and ecological roles. This knowledge will facilitate their further biotechnological exploitation and point the way towards development of pharmaceuticals and therapeutic applications.

Graphene Oxide Membranes with Heterogeneous Nanodomains for Efficient CO2 Separations

AbstractAchieving high membrane performance in terms of gas permeance and carbon dioxide selectivity is an important target in carbon capture. Aiming to manipulate the channel affinity towards CO2 to implement efficient separations, gas separation membranes containing CO2‐philic and non‐CO2‐philic nanodomains in the interlayer channels of graphene oxide (GO) were formed by intercalating poly(ethylene glycol) diamines (PEGDA). PEGDA reacts with epoxy groups on the GO surface, constructing CO2‐philic nanodomains and rendering a high sorption capacity, whereas unreacted GO surfaces give non‐CO2‐philic nanodomains, rendering low‐friction diffusion. Owing to the orderly stacking of nanochannels through cross‐linking and the heterogeneous nanodomains with moderate CO2 affinity, a GO‐PEGDA500 membrane exhibits a high CO2 permeance of 175.5 GPU and a CO2/CH4 selectivity of 69.5, which is the highest performance reported for dry‐state GO‐stacking membranes.

Graphene Oxide Membranes with Heterogeneous Nanodomains for Efficient CO2 Separations.

Achieving high membrane performance in terms of gas permeance and carbon dioxide selectivity is an important target in carbon capture. Aiming to manipulate the channel affinity towards CO2 to implement efficient separations, gas separation membranes containing CO2 -philic and non-CO2 -philic nanodomains in the interlayer channels of graphene oxide (GO) were formed by intercalating poly(ethylene glycol) diamines (PEGDA). PEGDA reacts with epoxy groups on the GO surface, constructing CO2 -philic nanodomains and rendering a high sorption capacity, whereas unreacted GO surfaces give non-CO2 -philic nanodomains, rendering low-friction diffusion. Owing to the orderly stacking of nanochannels through cross-linking and the heterogeneous nanodomains with moderate CO2 affinity, a GO-PEGDA500 membrane exhibits a high CO2 permeance of 175.5 GPU and a CO2 /CH4 selectivity of 69.5, which is the highest performance reported for dry-state GO-stacking membranes.

HBK-14 and HBK-15, triple 5-HT1A, 5-HT7 and 5-HT3 antagonists with potent antidepressant- and anxiolytic-like properties, increase seizure threshold in various seizure tests in mice

Most antidepressants lower seizure threshold, which might be due to the modulation of serotonergic neurotransmission. Here, we investigated the effects of two 5-HT1A, 5-HT7 and 5-HT3 antagonists, i.e., 1-(2-(2-(2,6-dimethylphenoxy)ethoxy)ethyl)-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-14) and 1-{2-[2-(2-chloro-6-methylphenoxy)ethoxy]ethyl}-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-15), with antidepressant- and anxiolytic-like properties, on seizure thresholds in three acute seizure tests, i.e., the intravenous pentylenetetrazole, maximal electroshock seizure threshold (MEST), and 6-Hz corneal stimulation test in mice. We also evaluated their affinity for voltage-gated sodium channels. Our results indicate that HBK-14 increased seizure thresholds in three seizure tests in mice, while HBK-15 was active in the MEST and 6-Hz tests. None of the compounds affected neuromuscular strength or motor coordination at active doses. We showed that both compounds had high affinity for voltage-dependent sodium channels, which combined with the influence on 5-HT1A, 5-HT7 and 5-HT3 receptors, might underlie their anticonvulsant effects. Since most antidepressants lower the seizure threshold, the fact that both compounds with potent antidepressant-like activity, increased or had no influence on seizure threshold is worth investigating.

Computational Tool for Fast in silico Evaluation of hERG K+ Channel Affinity.

The development of a novel comprehensive approach for the prediction of hERG activity is herein presented. Software Phase has been used to derive a 3D-QSAR model, employing as alignment rule a common pharmacophore built on a subset of 22 highly active compounds (threshold Ki: 50 nM) against hERG K+ channel. Five features comprised the pharmacophore: two aromatic rings (R1 and R2), one hydrogen-bond acceptor (A), one hydrophobic site (H), and one positive ionizable function (P). The sequential 3D-QSAR model developed with a set of 421 compounds (randomly divided in training and test set) yielded a test set (Q2) = 0.802 and proved to be predictive with respect to an external test set of 309 compounds that were not used to generate the model ( = 0.860). Furthermore, the model was submitted to an in silico validation for assessing the reliability of the approach, by applying a decoys set, evaluating the Güner and Henry score (GH) and the Enrichment Factor (EF), and by using the ROC curve analysis. The outcome demonstrated the high predictive power of the inclusive 3D-QSAR model developed for the hERG K+ channel blockers, confirming the fundamental validity of the chosen approach for obtaining a fast proprietary cardiotoxicity predictive tool to be employed for rationally designing compounds with reduced hERG K+ channel activity at the early steps of the drug discovery trajectory.

3D-SDAR modeling of hERG potassium channel affinity: A case study in model design and toxicophore identification

A dataset of 237 human Ether-à-go-go Related Gene (hERG) potassium channel inhibitors (180 of which were used for model building and validation, whereas 57 constituted the “true” external prediction set) collected from 22 literature sources was modeled by 3D-SDAR. To produce reliable and reproducible classification models for hERG blocking, the initial set of 180 chemicals was split into two subsets: a balanced modeling set consisting of 118 compounds and an unbalanced validation set comprised of 62 compounds. A PLS bagging-like algorithm written in Matlab was used to process the data and assign each compound to one of the two (hERG+ or hERG-) activity classes. The best predictive model evaluated on the basis of a fully randomized hold-out test set (comprising 20% of the modeling set) used 4 latent variables and a grid of 6ppm×6ppm×1Å in the C-C region, 6ppm×30ppm×1Å in the C-N region, and 30ppm×30ppm×1Å in the N-N region. An overall accuracy of 0.84 was obtained for both the hold-out test set and the validation set. Further, an external prediction set consisting of 57 drugs and drug derivatives was used to estimate the true predictive power of the reported 3D-SDAR model – a slight reduction of the overall accuracy down to 0.77 was observed. 3D-SDAR map of the most frequently occurring bins and their projection on the standard coordinate space of the chemical structures allowed identification of a three-center toxicophore composed of two aromatic rings and an amino group. A U test along the distance axis of the most frequently occurring 3D-SDAR bins was used to set the distance limits of the toxicophore. This toxicophore was found to be similar to an earlier reported phospholipidosis (PLD) toxicophore.

Effects of Benzothiazolamines on Voltage-Gated Sodium Channels.

Benzothiazole is a versatile fused heterocycle that aroused much interest in drug discovery as anticonvulsant, neuroprotective, analgesic, anti-inflammatory, antimicrobial, and anticancer. Two benzothiazolamines, riluzole and lubeluzole, are known blockers of voltage-gated sodium (Nav) channels. Riluzole is clinically used as a neuroprotectant in amyotrophic lateral sclerosis. Inhibition of Nav channels by riluzole is voltage-dependent due to preferential binding to inactivated sodium channels. Yet the drug exerts little use-dependent block, probably because it lacks protonable amine. One important property is riluzole ability to inhibit persistent Na+ currents, which likely contributes to its neuroprotective activity. Lubeluzole showed promising neuroprotective effects in animal stroke models, but failed to show benefits in acute ischemic stroke in humans. One important concern is its propensity to prolong the cardiac QT interval, due to hERG K+ channel block. Lubeluzole very potently inhibits Nav channels in a voltage- and use-dependent manner, due to its great preferential affinity for inactivated channels and the presence of a protonable amine group. Patch-clamp experiments suggest that the binding sites of both drugs overlap the local anesthetic receptor within the ion-conducting pathway. Riluzole and lubeluzole displayed very potent antimyotonic activity in a rat model of myotonia, a pathological skeletal muscle condition characterized by high-frequency runs of action potentials. Such results well support the repurposing of riluzole as an antimyotonic drug, allowing the launch of a pilot study in myotonic patients. Riluzole, lubeluzole, and new Nav channel blockers built on the benzothiazolamine scaffold will certainly continue to be investigated for possible clinical applications.

Protein arginine methylation facilitates KCNQ channel-PIP2 interaction leading to seizure suppression.

KCNQ channels are critical determinants of neuronal excitability, thus emerging as a novel target of anti-epileptic drugs. To date, the mechanisms of KCNQ channel modulation have been mostly characterized to be inhibitory via Gq-coupled receptors, Ca(2+)/CaM, and protein kinase C. Here we demonstrate that methylation of KCNQ by protein arginine methyltransferase 1 (Prmt1) positively regulates KCNQ channel activity, thereby preventing neuronal hyperexcitability. Prmt1+/- mice exhibit epileptic seizures. Methylation of KCNQ2 channels at 4 arginine residues by Prmt1 enhances PIP2 binding, and Prmt1 depletion lowers PIP2 affinity of KCNQ2 channels and thereby the channel activities. Consistently, exogenous PIP2 addition to Prmt1+/- neurons restores KCNQ currents and neuronal excitability to the WT level. Collectively, we propose that Prmt1-dependent facilitation of KCNQ-PIP2 interaction underlies the positive regulation of KCNQ activity by arginine methylation, which may serve as a key target for prevention of neuronal hyperexcitability and seizures.

Calmodulin and Fibroblast Growth Factor Homologous Factor Co-Regulation of the NaV1.5 Channel in LQT3 Mutations

Long QT syndrome type 3 (LQT3) results from mutations in the cardiac sodium channel NaV1.5 that increase the “late” Na+ current. Previous studies implicated the NaV1.5 auxiliary subunit apocalmodulin (apoCaM), a Ca2+ binding protein that interacts with the channel's cytosolic C-terminal domain (CTD), in the regulation of late Na+ current since a mutant channel unable to bind apoCaM displayed increased late current. Recent structural studies defined the precise apoCaM interaction sites on the CTD, which also binds a fibroblast growth factor homologous factor (FHF). How CaM influences the late Na+ current and whether the nearby FHF also regulates late current is unknown. Here, we queried the roles of both channel auxiliary subunits with biochemical and functional approaches by examining LQT3 mutations within the CTD.We found that: 1) FHF and CaM interaction with the NaV1.5 CTD is cooperative; 2) The LQT3 mutations E1901Q, Q1909R, and R1913H lower the affinity of the CTD for CaM; and 3) When expressed in HEK293 cells, all three mutant NaV1.5 channels had increased late Na+ current. We hypothesized that increased late current results from decreased affinity for CaM and/or the FHF, which we tested by re-measuring late current after overexpressing either CaM or FGF13, an FHF expressed in heart. Our rationale was that CaM overexpression would, by mass action, restore CaM binding to the mutant or that FHF expression, via its cooperative influence on CaM binding, would increase the mutant channel's affinity for CaM. Indeed, for all three mutants over-expression of an FHF or CaM decreased late current. Thus, we propose that CaM and an FHF co-operate to regulate NaV1.5 late Na+ current and that LQT3 mutations perturbing interaction of CaM or the FHF will enhance pathologic late Na+ current.

Scorpion toxins prefer salt solutions.

There is a wide variety of ion channel types with various types of blockers, making research in this field very complicated. To reduce this complexity, it is essential to study ion channels and their blockers independently. Scorpion toxins, a major class of blockers, are charged short peptides with high affinities for potassium channels. Their high selectivity and inhibitory properties make them an important pharmacological tool for treating autoimmune or nervous system disorders. Scorpion toxins typically have highly charged surfaces and-like other proteins-an intrinsic ability to bind ions (Friedman J Phys Chem B 115(29):9213-9223, 1996; Baldwin Biophys J 71(4):2056-2063, 1996; Vrbka et al. Proc Natl Acad Sci USA 103(42):15440-15444, 2006a; Vrbka et al. J Phys Chem B 110(13):7036-43, 2006b). Thus, their effects on potassium channels are usually investigated in various ionic solutions. In this work, computer simulations of protein structures were performed to analyze the structural properties of the key residues (i.e., those that are presumably involved in contact with the surfaces of the ion channels) of 12 scorpion toxins. The presence of the two most physiologically abundant cations, Na(+) and K(+), was considered. The results indicated that the ion-binding properties of the toxin residues vary. Overall, all of the investigated toxins had more stable structures in ionic solutions than in water. We found that both the number and length of elements in the secondary structure varied depending on the ionic solution used (i.e., in the presence of NaCl or KCl). This study revealed that the ionic solution should be chosen carefully before performing experiments on these toxins. Similarly, the influence of these ions should be taken into consideration in the design of toxin-based pharmaceuticals.

A web portal for in-silico action potential predictions

IntroductionMultiple cardiac ion channels are prone to block by pharmaceutical compounds, and this can have large implications for cardiac safety. The effect of a compound on individual ion currents can now be measured in automated patch clamp screening assays. In-silico action potential models are proposed as one way of predicting the integrated compound effects on whole-cell electrophysiology, to provide an improved indication of pro-arrhythmic risk. MethodsWe have developed open source software to run cardiac electrophysiology simulations to predict the overall effect of compounds that block IKr, ICaL, INa, IKs, IK1 and Ito to varying degrees, using a choice of mathematical electrophysiology models. To enable safety pharmacology teams to run and evaluate these simulations easily, we have also developed an open source web portal interface to this simulator. ResultsThe web portal can be found at https://chaste.cs.ox.ac.uk/ActionPotential. Users can enter details of compound affinities for ion channels in the form of IC50 or pIC50 values, run simulations, store the results for later retrieval, view summary graphs of the results, and export data to a spreadsheet format. DiscussionThis web portal provides a simple interface to reference versions of mathematical models, and well-tested state-of-the-art equation solvers. It provides safety teams easy access to the emerging technology of cardiac electrophysiology simulations for use in the drug-discovery process.

Validation of KCa3.1 Channel Nifedipine Interaction Site Predicted by Rosetta Modeling Method

Calcium-activated potassium channels, including the intermediate-conductance KCa3.1 channel and the related small-conductance KCa2 channels, monitor and regulate intracellular calcium and are ideal pharmacological targets for immunosuppression, fibroproliferative disorders, hypertension and various neurological diseases. However, the development of drugs specifically for these medically relevant channels faces serious challenges because there is no available crystal structure that could be used for structure-assisted drug design. We used the Kv1.2-Kv2.1 channel structure (pdb id: 2R9R) as a template to generate a homology model of the KCa3.1 channel transmembrane region using the Rosetta modeling method. Docking of small molecules that are known to block KCa3.1 channel currents using Rosetta generated structural models of channel - drug complexes that can be validated using experimental approaches. Here we report the validation of the binding sites for the dihydropyridine nifedipine predicted by Rosetta's lowest energy stimulation of the KCa3.1 channel complex. We used a combination of site-directed mutagenesis and electrophysiological patch-clamp recording to test the identified amino acid residues located between the pore lining S5 and S6 segment. Blocking of the KCa3.1 channel by nifedipine was reduced by altering the side chains at positions L209, T212 and V278 to either alanine or valine, with T212 the most affected residue. Bulkier side-chain change to phenylalanine confirmed T212 as the main interacting site for nifedipine without compromising the channel's affinity for TRAM-34. Thus, by correctly identified the interaction of the dihydropyridine nifedipine with the KCa3.1 channel complex, Rosetta models can be used to understand the molecular mechanism of action of KCa channel blockers and gating modulators and assist with drug design efforts.

Calculation of Cholesterol Binding Affinity for Pentameric Ligand-Gated Ion Channels

Cholesterol has been shown to play a critical role in the function of ion channels and many types of ion channels partition into cholesterol-rich membranes. Several Eukaryotic pentameric ligand-gated ion channels, including the nicotinic Acetylcholine Receptor and the GABA(A) receptor, are highly sensitive to the cholesterol content of reconstitution mixtures or native membranes, but the binding interactions between cholesterol and these receptors are largely uncharacterized. Since the mechanisms for modulation by cholesterol are likely to be at least partially through direct interactions via specific binding, cholesterol molecules may form an essential component of the native structure for some of these receptors. We recently proposed, based on the crystal structure of the glutamate-gated chloride channel (GluCl) from C. elegans solved in complex with the lipophilic ligand ivermectin, that cholesterol molecules could bind to these intersubunit sites in a pose analogous to that of ivermectin in the absence of other modulators, and tested this model in the homologous GABA(A) receptor using Molecular Dynamics (MD) simulations. Here we employ computational free energy calculation methods to provide a quantitative estimate of the binding affinity of cholesterol for these sites on GluCl and other pentameric ligand-gated ion channels, demonstrating that these sites are likely to be partially occupied at physiological concentrations of cholesterol.