Pharmacology Of Channels Research Articles

TASK Channels Pharmacology: New Challenges in Drug Design.

Rational drug design targeting ion channels is an exciting and always evolving research field. New medicinal chemistry strategies are being implemented to explore the wild chemical space and unravel the molecular basis of the ion channels modulators binding mechanisms. TASK channels belong to the two-pore domain potassium channel family and are modulated by extracellular acidosis. They are extensively distributed along the cardiovascular and central nervous systems, and their expression is up- and downregulated in different cancer types, which makes them an attractive therapeutic target. However, TASK channels remain unexplored, and drugs designed to target these channels are poorly selective. Here, we review TASK channels properties and their known blockers and activators, considering the new challenges in ion channels drug design and focusing on the implementation of computational methodologies in the drug discovery process.

Immunomagnetic cell separation is a suitable method for T cell electrophysiology and ion channel pharmacology

Abstract Immunomagnetic cell separation is a powerful tool of immunology, however the presence of beads on the cell surface might alter the electrophysiological and pharmacological parameters of the ion channels expressed in the separated cells. We addressed this issue by measuring the aforementioned properties of Kv1.3, the predominant voltage-gated K+ channel of activated T cells upon immunomagnetic separation. PBMCs from healthy donors were activated then CD4+ cells were separated with REAlease® CD4 Kit (Miltenyi Biotec Inc.). As control, we made stepwise removal of REAlease® antibody-bead complex, immunomagnetic negative separation and FACS. Purity and viability was determined by flow cytometry. Electrophysiology studies were made with patch-clamp technique in whole-cell configuration. The presence of bead-antibody complex statistically significantly increased the time constant (τact) characterizing the activation kinetics of the current as compared to negative selection (p=0.049) and antibody only (p=0.024) controls, however the increase in τact from ~0.5 ms to ~0.6 ms does not bear any biological significance. Other gating parameters of Kv1.3, such as inactivation time constant and midpoint of voltage-dependence of steady-state activation were unaffected by the beads. Moreover, the binding of a peptide (ChTx) and a small organic molecule (TEA) inhibitor to Kv1.3 was oblivious to the presence of beads. In addition, the electrical capacitance of the cell membrane was not altered when beads were attached to the cells. Based on our results we conclude that the application of immunomagnetic cell separation is completely suitable to electrophysiology studies of immune cells.

Role of SK channel activation in determining the action potential configuration in freshly isolated human atrial myocytes from the SKArF study

Inhibition of SK channel function is being pursued in animal models as a possible therapeutic approach to treat atrial fibrillation (AF). However, the pharmacology of SK channels in human atria is unclear. SK channel function is inhibited by both apamin and UCL1684, with the former discriminating between SK channel subtypes. In this proof-of-principle study, the effects of apamin and UCL1684 on right atrial myocytes freshly isolated from patients in sinus rhythm undergoing elective cardiac surgery were investigated. Outward current evoked from voltage clamped human atrial myocytes was reduced by these two inhibitors of SK channel function. In contrast, membrane current underlying the atrial action potential was affected significantly only by UCL1684 and not by apamin. This pharmacology mirrors that observed in mouse atria, suggesting that mammalian atria possess two populations of SK channels, with only one population contributing to the action potential waveform. Immuno-visualization of the subcellular localization of SK2 and SK3 subunits showed a high degree of colocalization, consistent with the formation of heteromeric SK2/SK3 channels. These data reveal that human atrial myocytes express two SK channel subtypes, one exhibiting an unusual pharmacology. These channels contribute to the atrial action potential waveform and might be a target for novel therapeutic approaches to treat supraventricular arrhythmic conditions such as atrial fibrillation.

Use of calcium channel blockers in cardiovascular disease

Calcium channel blockers (CCBs) are widely used agents in the management of a number of cardiovascular indications, including hypertension, angina and supraventricular tachycardias. By inhibiting the movement of calcium into the cytoplasm via transmembrane calcium channels, they reduce vascular and myocardial contractility, and modulate the activity of pacemaker cells. There are important differences between the different types of calcium channel blockers, and a sound understanding of their mechanism of action is required to promote their safe, effective use. This article briefly describes calcium channel pharmacology, the mechanism of action of CCBs and their place in the therapy of cardiovascular disease.

Whole-Cell Patch Clamp Recordings from Oligodendrocyte Lineage Cells in Brain Slices.

The whole-cell configuration of the patch-clamp technique is widely used to study electrically active cells and passive membrane properties, as well as the properties and pharmacology of ion channels, neurotransmitter receptors, and electrogenic transporters, in almost any cell type. In the brain, in addition to neurons, oligodendrocyte precursor cells (OPCs) that give rise to myelinating oligodendrocytes (OLs) are also excitable. Electrophysiological techniques provide the main tool for the thorough investigation of the electrogenic capacity of such cell types. Although there are many published protocols for whole-cell recordings, there are very few that touch upon the electrophysiological characteristics of oligodendrocyte lineage cells. Here we provide a detailed methodology for how to acquire and analyze whole-cell recordings from excitable cells, with a focus on oligodendrocyte lineage cells. We provide a protocol on how to successfully identify OPCs and OLs in brain slices, either with the use of transgenic animal models or through morphological and electrophysiological profiling. The method described can also be easily adopted for whole-cell recordings from oligodendrocyte lineage cells in vitro.

Ion Channel Pharmacology for Pain Modulation.

A large series of different ion channels have been identified and investigated as potential targets for new medicines for the treatment of a variety of human diseases, including pain. Among these channels, the voltage gated calcium channels (VGCC) are inhibited by drugs for the treatment of migraine, neuropathic pain or intractable pain. Transient receptor potential (TRP) channels are emerging as important pain transducers as they sense low pH media or oxidative stress and other mediators and are abundantly found at sites of inflammation or tissue injury. Low pH may also activate acid sensing ion channels (ASIC) and mechanical forces stimulate the PIEZO channels. While potent agonists of TRP channels due to their desensitizing action on pain transmission are used as topical applications, the potential of TRP antagonists as pain therapeutics remains an exciting field of investigation. The study of ASIC or PIEZO channels in pain signaling is in an early stage, whereas antagonism of the purinergic P2X3 channels has been reported to provide beneficial effects in chronic intractable cough. The present chapter covers these intriguing channels in great detail, highlighting their diverse mechanisms and broad potential for therapeutic utility.

Analgesic Effects of the GIRK Activator, VU0466551, Alone and in Combination with Morphine in Acute and Persistent Pain Models.

G protein-gated inwardly rectifying potassium (GIRK) channels are potassium-selective ion channels. As their name suggests, GIRK channels are effectors of Gi/o G protein-couple receptors whereby activation of these GPCRs leads to increased GIRK channel activity resulting in decreased cellular excitability. In this way, GIRK channels play diverse roles in physiology as effectors of Gi/o-coupled GPCRs: peacemaking in the heart rate, modulation of hormone secretion in endocrine tissues, as well as numerous CNS functions including learning, memory, and addiction/reward. Notably, GIRK channels are widely expressed along the spinothalamic tract and are positioned to play roles in both ascending and descending pain pathways. More notably, GIRK channel knockout and knock-down studies have found that GIRK channels play a major role in the action of opioid analgesics which act predominantly through Gi/o-coupled, opioid-activated GPCRs (e.g., μ-opioid receptors). Recent advances in GIRK channel pharmacology have led to the development of small molecules that directly and selectively activate GIRK channels. Based on research implicating the involvement of GIRK channels in pain pathways and as effectors of opioid analgesics, we conducted a study to determine whether direct pharmacological activation of GIRK channels could produce analgesic efficacy and/or augment the analgesic efficacy morphine, an opioid receptor agonist capable of activating μ-opioid receptors as well as other opioid receptor subtypes. In the present study, we demonstrate that the small-molecule GIRK activator, VU0466551, has analgesic effects when dosed alone or in combination with submaximally effective doses of morphine.

Current Concepts and Perspectives on Connexin43: A Mini Review.

Connexins are a family of gap junction proteins widely distributed in human organs and tissues. Gap junctions are organized systems of intercellular protein channels that allow the exchange of ions, chemical signals, and energy substrates between two adjacent cells. Connexin43 (Cx43) is the most abundant isoform of connexins in the heart which play an important role in myocardium disease. Numerous studies have shown that Cx43 was involved in tumor migration and invasion by mediating gap junctions between tumor cells and normal cells. Changes in the expression and distribution of Cx43 contribute to heart disease and cancer. This review discusses current knowledge on the functional and structural abnormalities in Cx43 associated with heart disease and cancer, aiming to highlight the importance of this connexin as an emerging therapeutic target. Here, the current knowledge on the pharmacology of Gap Junction Channels and Hemichannels were also summarized. Finally, we propose that these knowledges can be exploited to identify new diagnostic and effective therapeutic approaches for ischemic heart disease and cancer.

Temperature effect on hERG channel pharmacology measured by using the Qube automated patch clamp system

Temperature effect on hERG channel pharmacology measured by using the Qube automated patch clamp system

Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel NaV1.7.

Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate NaV1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.

Assessment of an In Silico Mechanistic Model for Proarrhythmia Risk Prediction Under the CiPA Initiative.

The International Council on Harmonization (ICH) S7B and E14 regulatory guidelines are sensitive but not specific for predicting which drugs are pro‐arrhythmic. In response, the Comprehensive In Vitro Proarrhythmia Assay (CiPA) was proposed that integrates multi‐ion channel pharmacology data in vitro into a human cardiomyocyte model in silico for proarrhythmia risk assessment. Previously, we reported the model optimization and proarrhythmia metric selection based on CiPA training drugs. In this study, we report the application of the prespecified model and metric to independent CiPA validation drugs. Over two validation datasets, the CiPA model performance meets all pre‐specified measures for ranking and classifying validation drugs, and outperforms alternatives, despite some in vitro data differences between the two datasets due to different experimental conditions and quality control procedures. This suggests that the current CiPA model/metric may be fit for regulatory use, and standardization of experimental protocols and quality control criteria could increase the model prediction accuracy even further.

Targeting Mitochondrial Ion Channels to Fight Cancer.

In recent years, several experimental evidences have underlined a new role of ion channels in cancer development and progression. In particular, mitochondrial ion channels are arising as new oncological targets, since it has been proved that most of them show an altered expression during tumor development and the pharmacological targeting of some of them have been demonstrated to be able to modulate cancer growth and progression, both in vitro as well as in vivo in pre-clinical mouse models. In this scenario, pharmacology of mitochondrial ion channels would be in the near future a new frontier for the treatment of tumors. In this review, we discuss the new advances in the field, by focusing our attention on the improvements in new drug developments to target mitochondrial ion channels.

Cardiovascular pharmacology of K2P17.1 (TASK-4, TALK-2) two-pore-domain K+ channels.

K2P17.1 (TASK-4, TALK-2) potassium channels are expressed in the heart and represent potential targets for pharmacological management of atrial and ventricular arrhythmias. Reduced K2P17.1 expression was found in atria and ventricles of heart failure (HF) patients. Modulation of K2P17.1 currents by antiarrhythmic compounds has not been comprehensively studied to date. The objective of this study was to investigate acute effects of clinically relevant antiarrhythmic drugs on human K2P17.1 channels to provide a more complete picture of K2P17.1 electropharmacology. Whole-cell patch clamp and two-electrode voltage clamp electrophysiology was employed to study human K2P17.1 channel pharmacology. K2P17.1 channels expressed in Xenopus laevis oocytes were screened for sensitivity to antiarrhythmic drugs, revealing significant activation by propafenone (+ 296%; 100μM), quinidine (+ 58%; 100μM), mexiletine (+ 21%; 100μM), propranolol (+ 139%; 100μM), and metoprolol (+ 17%; 100μM) within 60min. In addition, the currents were inhibited by amiodarone (- 13%; 100μM), sotalol (- 10%; 100μM), verapamil (- 21%; 100μM), and ranolazine (- 8%; 100μM). K2P17.1 channels were not significantly affected by ajmaline and carvedilol. Concentration-dependent K2P17.1 activation by propafenone was characterized in more detail. The onset of activation was fast, and current-voltage relationships were not modulated by propafenone. K2P17.1 activation was confirmed in mammalian Chinese hamster ovary cells, revealing 7.8-fold current increase by 100μM propafenone. Human K2P17.1 channels were sensitive to multiple antiarrhythmic drugs. Differential pharmacological regulation of repolarizing K2P17.1 background K+ channels may be employed for personalized antiarrhythmic therapy.

Cardiac K2P13.1 (THIK-1) two-pore-domain K+ channels: Pharmacological regulation and remodeling in atrial fibrillation

Cardiac two-pore-domain potassium (K2P) channels have been proposed as novel antiarrhythmic targets. K2P13.1 (THIK-1) channels are expressed in the human heart, and atrial K2P13.1 levels are reduced in patients with atrial fibrillation (AF) or heart failure. The first objective of this study was to investigate acute effects of antiarrhythmic drugs on human K2P13.1 currents. Second, we assessed atrial K2P13.1 remodeling in AF pigs to validate the porcine model for future translational evaluation of K2P13.1-based antiarrhythmic concepts. K2P13.1 protein expression was studied in domestic pigs during AF induced by atrial burst pacing. AF was associated with 66% reduction of K2P13.1 levels in the right atrium at 21-day follow-up. Voltage clamp electrophysiology was employed to elucidate human K2P13.1 channel pharmacology in Xenopus oocytes. Propafenone (−26%; 100 μM), mexiletine (−75%; 1.5 mM), propranolol (−38%; 200 μM), and lidocaine (−59%; 100 μM) significantly inhibited K2P13.1 currents. By contrast, K2P13.1 channels were not markedly affected by quinidine, carvedilol, metoprolol, amiodarone and verapamil. Concentration-dependent K2P13.1 blockade by mexiletine occurred rapidly with membrane depolarization and was frequency-independent. Mexiletine reduced K2P13.1 open rectification properties and shifted current-voltage relationships towards more negative potentials. In conclusion, atrial expression and AF-associated downregulation of K2P13.1 channels in a porcine model resemble human findings and support a general role for K2P13.1 in AF pathophysiology. K2P13.1 current sensitivity to antiarrhythmic drugs provides a starting point for further development of an emerging antiarrhythmic paradigm.

Investigation of hERG1b Influence on hERG Channel Pharmacology at Physiological Temperature

Objective: To compare the inhibitory potencies of selected drugs (chloroquine, fluoxetine, cisapride, and ebastine [EBA]) on human Ether-a-go-go-Related Gene (hERG) potassium channel current carried by either hERG1a or co-expressed hERG 1a/1b channel isoforms. Materials and Methods: Measurements of hERG current (IhERG) were made at 37°C from HEK-293 cells expressing either the hERG1a isoform or co-expressing hERG1a and 1b isoforms. A standard “square” waveform voltage protocol was used to elicit IhERG, and tail current measurements were used to construct concentration-response relations for each drug. Results: For fluoxetine, cisapride, and chloroquine, the observed potencies of inhibition of IhERGwere similar between hERG1 and 1a/1b expression conditions. Further experiments in which the hERG1b isoform was expressed alone also failed to show different potencies from hERG1a for these drugs. Fluoxetine was also tested at room temperature and showed similar potencies against hERG 1a and 1a/1b. EBA was more potent against hERG1a than hERG1a/1b with respective half maximal inhibitory concentration (IC50) values of 32 nM ( 95% confidence interval [CI] 24 nM–43 nM) and 185 nM (CI 114 nM–304 nM), a 5.8-fold difference. At ambient temperature, EBA was also more potent against hERG1a than 1a/1b, with a 2.4-fold difference in IC50. Conclusion: Comparison of these findings with prior planar patch-clamp data suggests that automated patch-clamp data on hERG1a/1b versus hERG 1a at ambient temperature cannot automatically be extrapolated to manual patch clamp at 37°C. The results with EBA highlight that, during hERG screening of novel drugs, there is a case for promising candidates to incorporate some measurements on hERG1a/1b as well as hERG1a channels.

Co-expression of β Subunits with the Voltage-Gated Sodium Channel NaV1.7: the Importance of Subunit Association and Phosphorylation and Their Effects on Channel Pharmacology and Biophysics.

The voltage-gated sodium ion channel NaV1.7 is crucial in pain signaling. We examined how auxiliary β2 and β3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human NaV1.7α subunit was co-expressed with either β2 or β3 subunits in HEK-293 cells. The β2 subunits and the NaV1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the β2 subunits did not change the biophysical properties of the channel. In contrast, β3 subunit was clearly associated with NaV1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated β3 subunit was associated with the NaV1.7α. Electrophysiological characterisation revealed that the β3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the β3 subunit reduced the susceptibility of NaV1.7α to several sodium channel blockers. In addition, we assessed the functional effect of NaV1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of β subunits with NaV1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with β2. The β3 subunit significantly influences the function of NaV1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of NaV1.7 channel in pain signaling.

Atypical pharmacology of schistosome TRPA1-like ion channels.

Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease estimated to affect over 200 million people worldwide. Praziquantel is the only antischistosomal currently available for treatment, and there is an urgent need for new therapeutics. Ion channels play key roles in physiology and are targets for many anthelmintics, yet only a few representatives have been characterized in any detail in schistosomes and other parasitic helminths. The transient receptor potential (TRP) channel superfamily comprises a diverse family of non-selective cation channels that play key roles in sensory transduction and a wide range of other functions. TRP channels fall into several subfamilies. Members of both the TRPA and TRPV subfamilies transduce nociceptive and inflammatory signals in mammals, and often also respond to chemical and thermal signals. We previously showed that although schistosomes contain no genes predicted to encode TRPV channels, TRPV1-selective activators such as capsaicin and resiniferatoxin elicit dramatic hyperactivity in adult worms and schistosomula. Surprisingly, this response requires expression of a S. mansoni TRPA1-like orthologue (SmTRPA). Here, we show that capsaicin induces a rise in intracellular Ca2+ in mammalian cells expressing either SmTRPA or a S. haematobium TRPA1 orthologue (ShTRPA). We also test SmTRPA and ShTRPA responses to various TRPV1 and TRPA1 modulators. Interestingly, in contrast to SmTRPA, ShTRPA is not activated by the TRPA1 activator AITC (allyl isothiocyanate), nor do S. haematobium adult worms respond to this compound, a potentially intriguing species difference. Notably, 4-hydroxynonenal (4-HNE), a host-derived, inflammatory product that directly activates mammalian TRPA1, also activates both SmTRPA and ShTRPA. Our results point to parasite TRPA1-like channels which exhibit atypical, mixed TRPA1/TRPV1-like pharmacology, and which may also function to transduce endogenous host signals.

A Thallium-Based Screening Procedure to Identify Molecules That Modulate the Activity of Ca2+-Activated Monovalent Cation-Selective Channels

TRPM5 functions as a calcium-activated monovalent cation-selective ion channel and is expressed in a variety of cell types. Dysfunction of this type of channel has been recently implied in cardiac arrhythmias, diabetes, and other pathologies. Therefore, a growing interest has emerged to develop the pharmacology of these ion channels. We optimized a screening assay based on the thallium flux through the TRPM5 channel and a fluorescent thallium dye as a probe for channel activity. We show that this assay is capable of identifying molecules that inhibit or potentiate calcium-activated monovalent cation-selective ion channels.

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purifiedhEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

Munc13-3 Is Required for the Developmental Localization of Ca2+ Channels to Active Zones and the Nanopositioning of Cav2.1 Near Release Sensors

Spatial relationships between Cav channels and release sensors at active zones (AZs) are a major determinant of synaptic fidelity. They are regulated developmentally, but the underlying molecular mechanisms are largely unclear. Here, we show that Munc13-3 regulates the density of Cav2.1 and Cav2.2 channels, alters the localization of Cav2.1, and is required for the development of tight, nanodomain coupling at parallel-fiber AZs. We combined EGTA application and Ca2+-channel pharmacology in electrophysiological and two-photon Ca2+ imaging experiments with quantitative freeze-fracture immunoelectron microscopy and mathematical modeling. We found that a normally occurring developmental shift from release being dominated by Ca2+ influx through Cav2.1 and Cav2.2 channels with domain overlap and loose coupling (microdomains) to a nanodomain Cav2.1 to sensor coupling is impaired in Munc13-3-deficient synapses. Thus, at AZs lacking Munc13-3, release remained triggered by Cav2.1 and Cav2.2 microdomains, suggesting a critical role of Munc13-3 in the formation of release sites with calcium channel nanodomains.