KCNQ1 is an essential mediator of the sex-dependent perception of moderate cold temperatures.

Low temperatures and cooling agents like menthol induce cold sensation by activating the peripheral cold receptors TRPM8 and TRPA1, cation channels belonging to the TRP channel family, while the reduction of potassium currents provides an additional and/or synergistic mechanism of cold sensation. Despite extensive studies over the past decades to identify the molecular receptors that mediate thermosensation, cold sensation is still not fully understood and many cold-sensitive peripheral neurons do not express the well-established cold sensor TRPM8. We found that the voltage-gated potassium channel KCNQ1 (Kv7.1), which is defective in cardiac LQT1 syndrome, is, in addition to its known function in the heart, a highly relevant and sex-specific sensor of moderately cold temperatures. We found that KCNQ1 is expressed in skin and dorsal root ganglion neurons, is sensitive to menthol and cooling agents, and is highly sensitive to moderately cold temperatures, in a temperature range at which TRPM8 is not thermosensitive. C-fiber recordings from KCNQ1-/- mice displayed altered action potential firing properties. Strikingly, only male KCNQ1-/- mice showed substantial deficits in cold avoidance at moderately cold temperatures, with a strength of the phenotype similar to that observed in TRPM8-/- animals. While sex-dependent differences in thermal sensitivity have been well documented in humans and mice, KCNQ1 is the first gene reported to play a role in sex-specific temperature sensation. Moreover, we propose that KCNQ1, together with TRPM8, is a key instrumentalist that orchestrates the range and intensity of cold sensation.

Chemical, pharmacodynamic and pharmacokinetic characterization of the GluN2B receptor antagonist 3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol - starting point for PET tracer development.

GluN2B-NMDA receptors play a key role in several neurological and neurodegenerative disorders. In order to develop novel negative allosteric GluN2B-NMDA receptor modulators, the concept of conformational restriction was pursued, i.e. the flexible aminoethanol substructure of ifenprodil was embedded into a more rigid tetrahydro-3-benzazepine system. The resulting tetrahydro-3-benzazepine-1,7-diol (±)-2 (WMS-1410) showed promising receptor affinity in receptor binding studies (K i = 84 nM) as well as pharmacological activity in two-electrode-voltage-clamp experiments (IC 50 = 116 nM) and in cytoprotective assays (IC 50 = 18.5 nM). The interactions of (R)-2 with the ifenprodil binding site of GluN2B-NMDA receptors were analyzed on the molecular level and the "foot-in-the-door" mechanism was developed. Due to promising pharmacokinetic parameters (logD7.4 = 1.68, plasma protein binding of 76-77%, sufficient metabolic stability) F-substituted analogs were prepared and evaluated as tracers for positron emission tomography (PET). Both fluorine-18-labeled PET tracers [18F]11 and [18F]15 showed high brain uptake, specific accumulation in regions known for high GluN2B-NMDA receptor expression, but no interactions with σ 1 receptors. Radiometabolites were not observed in the brain. Both PET tracers might be suitable for application in humans.

Characterization of Kv1.2-mediated outward current in TRIP8b-deficient mice.

Tonic current through hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels is influencing neuronal firing properties and channel function is strongly influenced by the brain-specific auxiliary subunit tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Since Kv1.2 channels and TRIP8b were also suggested to interact, we assessed brain Kv1.2 mRNA and protein expression as well as the reduction of K+ outward currents by Kv1.2-blocking compounds (Psora-4; tityustoxin-Kα, TsTX-Kα) in different brain areas of TRIP8b-deficient (TRIP8b -/- ) compared to wildtype (WT) mice. We found that transcription levels of Kv1.2 channels were not different between genotypes. Furthermore, Kv1.2 current amplitude was not affected upon co-expression with TRIP8b in oocytes. However, Kv1.2 immunofluorescence was stronger in dendritic areas of cortical and hippocampal neurons. Furthermore, the peak net outward current was increased and the inactivation of the Psora-4-sensitive current component was less pronounced in cortical neurons in TRIP8b -/- mice. In current clamp recordings, application of TsTX increased the excitability of thalamocortical (TC) neurons with increased number of elicited action potentials upon step depolarization. We conclude that TRIP8b may not preferentially influence the amplitude of current through Kv1.2 channels but seems to affect current inactivation and channel localization. In TRIP8b -/- a compensatory upregulation of other Kv channels was observed.

A novel NMDA receptor test model based on hiPSC-derived neural cells.

N-Methyl-D-aspartate receptors (NMDARs) are central for learning and information processing in the brain. Dysfunction of NMDARs can play a key role in the pathogenesis of neurodegeneration and drug addiction. The development of selective NMDAR modulators represents a promising strategy to target these diseases. Among such modulating compounds are ifenprodil and its 3-benzazepine derivatives. Classically, the effects of these NMDAR modulators have been tested by techniques like two-electrode voltage clamp (TEVC), patch clamp, or fluorescence-based assays. However, testing their functional effects in complex human systems requires more advanced approaches. Here, we established a human induced pluripotent stem cell-derived (hiPSC-derived) neural cell system and proved its eligibility as a test system for investigating NMDAR modulators and pharmaceutical effects on human neurons.

The second PI(3,5)P2 binding site in the S0 helix of KCNQ1 stabilizes PIP2-at the primary PI1 site with potential consequences on intermediate-to-open state transition.

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.

Validation of TREK1 ion channel activators as an immunomodulatory and neuroprotective strategy in neuroinflammation.

Modulation of two-pore domain potassium (K2P) channels has emerged as a novel field of therapeutic strategies as they may regulate immune cell activation and metabolism, inflammatory signals, or barrier integrity. One of these ion channels is the TWIK-related potassium channel 1 (TREK1). In the current study, we report the identification and validation of new TREK1 activators. Firstly, we used a modified potassium ion channel assay to perform high-throughput-screening of new TREK1 activators. Dose-response studies helped to identify compounds with a high separation between effectiveness and toxicity. Inside-out patch-clamp measurements of Xenopus laevis oocytes expressing TREK1 were used for further validation of these activators regarding specificity and activity. These approaches yielded three substances, E1, B3 and A2 that robustly activate TREK1. Functionally, we demonstrated that these compounds reduce levels of adhesion molecules on primary human brain and muscle endothelial cells without affecting cell viability. Finally, we studied compound A2 via voltage-clamp recordings as this activator displayed the strongest effect on adhesion molecules. Interestingly, A2 lacked TREK1 activation in the tested neuronal cell type. Taken together, this study provides data on novel TREK1 activators that might be employed to pharmacologically modulate TREK1 activity.

Negative allosteric modulators of NMDA receptors with GluN2B subunit: synthesis of ß-aminoalcohols by epoxide opening and subsequent rearrangement.

In order to obtain novel antagonists of GluN2B subunit containing NMDA receptors, aryloxiranes were opened with benzylpiperidines. Phenyloxiranes 6 and (indazolyl)oxirane 15 were opened regioselectively at the position bearing the aryl moiety. Reaction of the resulting ß-aminoalcohols 7 and 16 with carboxylic acids under Mitsunobu conditions (DIAD, PPh3) led to rearrangement and after ester hydrolysis to the regioisomeric ß-aminoalcohols 9 and 18. This strategy allows the synthesis of amino-ifenprodil 12 as well using phthalimide in the Mitsunobu reaction. Unexpectedly, the isomeric (indazolyl)oxirane 21 reacted with benzylpiperidines to afford both regioisomeric ß-aminoalcohols 22 and 23. In radioligand receptor binding studies, the indazolyl derivative 18a, which can be regarded as indazole bioisostere of ifenprodil, showed high GluN2B affinity (Ki = 31 nM). Replacement of the benzylic OH moiety of ifenprodil by the NH2 moiety in amino-ifenprodil 12 also resulted in low nanomolar GluN2B affinity (Ki = 72 nM). In TEVC experiments, 18a inhibited the ion flux to the same extent as ifenprodil proving that the phenol of ifenprodil can be replaced bioisosterically by an indazole ring maintaining affinity and inhibitory activity. Whereas 10-fold selectivity was found for the ifenprodil binding site over σ1 receptors, only low preference for the GluN2B receptor over σ2 receptors was detected. The log D7.4 value of 18a (log D7.4 = 2.08) indicates promising bioavailability.

Virus-induced inhibition of cardiac pacemaker channel HCN4 triggers bradycardia in human-induced stem cell system.

The enterovirus Coxsackievirus B3 (CVB3) is known to be a major source for the development of cardiac dysfunctions like viral myocarditis (VMC) and dilatative cardiomyopathy (DCM), but also results in bradycardia and fatal cardiac arrest. Besides clinical reports on bradycardia and sudden cardiac death, very little is known about the influence of CVB3 on the activity of human cardiac pacemaker cells. Here, we address this issue using the first human induced pluripotent stem cell (hiPSC)-derived pacemaker-like cells, in which the expression of a transgenic non-infectious variant of CVB3 can be controlled dose- and time-dependently. We found that CVB3 drastically changed hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) distribution and function in hiPSC-derived pacemaker-like tissue. In addition, using HCN4 cell expression systems, we found that HCN4 currents were decreased with altered voltage dependency of activation when CVB3 was expressed. Increased autophagosome formation and autophagosomal HCN4 insertion was observed in hiPSC-derived pacemaker-like cells under CVB3 expression as well. Individual effects of single, non-structural CVB3 proteins were analyzed and demonstrated that CVB3 proteins 2C and 3A had the most robust effect on HCN4 activity. Treatment of cells with the Rab7 inhibitor CID 106770 or the CVB3-3A inhibitor GW5074 led to the recovery of the cytoplasmatic HCN4 accumulation into a healthy appearing phenotype, indicating that malfunctioning Rab7-directed autophagosome transport is involved in the disturbed, cytoplasmatic HCN4 accumulation in CVB3-expressing human pacemaker-like cells. Summarizing, the enterovirus CVB3 inhibits human cardiac pacemaker function by reducing the pacemaker channel plasma membrane density, an effect that can be corrected by pharmacological intervention of endocytic vesicle trafficking.

Activation of non-classical NMDA receptors by glycine impairs barrier function of brain endothelial cells.

Blood-brain barrier (BBB) integrity is necessary to maintain homeostasis of the central nervous system (CNS). NMDA receptor (NMDAR) function and expression have been implicated in BBB integrity. However, as evidenced in neuroinflammatory conditions, BBB disruption contributes to immune cell infiltration and propagation of inflammatory pathways. Currently, our understanding of the pathophysiological role of NMDAR signaling on endothelial cells remains incomplete. Thus, we investigated NMDAR function on primary mouse brain microvascular endothelial cells (MBMECs). We detected glycine-responsive NMDAR channels, composed of functional GluN1, GluN2A and GluN3A subunits. Importantly, application of glycine alone, but not glutamate, was sufficient to induce NMDAR-mediated currents and an increase in intracellular Ca2+ concentrations. Functionally, glycine-mediated NMDAR activation leads to loss of BBB integrity and changes in actin distribution. Treatment of oocytes that express NMDARs composed of different subunits, with GluN1 and GluN3A binding site inhibitors, resulted in abrogation of NMDAR signaling as measured by two-electrode voltage clamp (TEVC). This effect was only detected in the presence of the GluN2A subunits, suggesting the latter as prerequisite for pharmacological modulation of NMDARs on brain endothelial cells. Taken together, our findings argue for a novel role of glycine as NMDAR ligand on endothelial cells shaping BBB integrity.

Optimized synthesis and pharmacological evaluation of HCN channel inhibitor EC18.

HCN4 channels are considered to be a promising target for cardiac pathologies, epilepsy, and multiple sclerosis. However, there are no subtype-selective HCN channel blockers available, and only a few compounds are reported to display subtype preferences, one of which is EC18 (cis-1). Herein, we report the optimized synthetic route for the preparation of EC18 and its evaluation in three different pharmacological models, allowing us to assess its activity on cardiac function, thalamocortical neurons, and immune cells.

Knockout of the Cardiac Transcription Factor NKX2-5 Results in Stem Cell-Derived Cardiac Cells with Typical Purkinje Cell-like Signal Transduction and Extracellular Matrix Formation.

The human heart controls blood flow, and therewith enables the adequate supply of oxygen and nutrients to the body. The correct function of the heart is coordinated by the interplay of different cardiac cell types. Thereby, one can distinguish between cells of the working myocardium, the pace-making cells in the sinoatrial node (SAN) and the conduction system cells in the AV-node, the His-bundle or the Purkinje fibres. Tissue-engineering approaches aim to generate hiPSC-derived cardiac tissues for disease modelling and therapeutic usage with a significant improvement in the differentiation quality of myocardium and pace-making cells. The differentiation of cells with cardiac conduction system properties is still challenging, and the produced cell mass and quality is poor. Here, we describe the generation of cardiac cells with properties of the cardiac conduction system, called conduction system-like cells (CSLC). As a primary approach, we introduced a CrispR-Cas9-directed knockout of the NKX2-5 gene in hiPSC. NKX2-5-deficient hiPSC showed altered connexin expression patterns characteristic for the cardiac conduction system with strong connexin 40 and connexin 43 expression and suppressed connexin 45 expression. Application of differentiation protocols for ventricular- or SAN-like cells could not reverse this connexin expression pattern, indicating a stable regulation by NKX2-5 on connexin expression. The contraction behaviour of the hiPSC-derived CSLCs was compared to hiPSC-derived ventricular- and SAN-like cells. We found that the contraction speed of CSLCs resembled the expected contraction rate of human conduction system cells. Overall contraction was reduced in differentiated cells derived from NKX2-5 knockout hiPSC. Comparative transcriptomic data suggest a specification of the cardiac subtype of CSLC that is distinctly different from ventricular or pacemaker-like cells with reduced myocardial gene expression and enhanced extracellular matrix formation for improved electrical insulation. In summary, knockout of NKX2-5 in hiPSC leads to enhanced differentiation of cells with cardiac conduction system features, including connexin expression and contraction behaviour.

The Special One: Architecture, Physiology and Pharmacology of the TRESK Channel.

The TWIK-related spinal cord K+ channel (TRESK) is part of the two-pore domain K+ channel family (K2P), which are also called leak potassium channels. As indicated by the channel family name, TRESK conducts K+ ions along the concentration gradient in a nearly voltage-independent manner leading to lowered membrane potentials. Although functional and pharmacological similarities exist, TRESK shows low sequence identity with other K2P channels. Moreover, the channel possesses several unique features such as its sensitivity to intracellular Ca2+ ions, that are not found in other K2P channels. High expression rates are found in immune-associated and neuronal cells, especially in sensory neurons of the dorsal root and trigeminal ganglia. As a consequence of the induced hyperpolarization, TRESK influences neuronal firing, the release of inflammatory mediators and the proliferation of distinct immune cells. Consequently, this channel might be a suitable target for pharmacological intervention in migraine, epilepsy, neuropathic pain or distinct immune diseases. In this review, we summarize the biochemical and biophysical properties of TRESK channels as well as their sensitivity to different known compounds. Furthermore, we give a structured overview about the physiological and pathophysiological impact of TRESK, that render the channel as an interesting target for specific drug development.

Discrete spatio-temporal regulation of tyrosine phosphorylation directs influenza A virus M1 protein towards its function in virion assembly.

Small RNA viruses only have a very limited coding capacity, thus most viral proteins have evolved to fulfill multiple functions. The highly conserved matrix protein 1 (M1) of influenza A viruses is a prime example for such a multifunctional protein, as it acts as a master regulator of virus replication whose different functions have to be tightly regulated. The underlying mechanisms, however, are still incompletely understood. Increasing evidence points towards an involvement of posttranslational modifications in the spatio-temporal regulation of M1 functions. Here, we analyzed the role of M1 tyrosine phosphorylation in genuine infection by using recombinant viruses expressing M1 phosphomutants. Presence of M1 Y132A led to significantly decreased viral replication compared to wildtype and M1 Y10F. Characterization of phosphorylation dynamics by mass spectrometry revealed the presence of Y132 phosphorylation in M1 incorporated into virions that is most likely mediated by membrane-associated Janus kinases late upon infection. Molecular dynamics simulations unraveled a potential phosphorylation-induced exposure of the positively charged linker domain between helices 4 and 5, supposably acting as interaction platform during viral assembly. Consistently, M1 Y132A showed a defect in lipid raft localization due to reduced interaction with viral HA protein resulting in a diminished structural stability of viral progeny and the formation of filamentous particles. Importantly, reduced M1-RNA binding affinity resulted in an inefficient viral genome incorporation and the production of non-infectious virions that interferes with virus pathogenicity in mice. This study advances our understanding of the importance of dynamic phosphorylation as a so far underestimated level of regulation of multifunctional viral proteins and emphasizes the potential feasibility of targeting posttranslational modifications of M1 as a novel antiviral intervention.

Phenol-Benzoxazolone bioisosteres: Synthesis and biological evaluation of tricyclic GluN2B-selective N-methyl- d-aspartate receptor antagonists.

Tricyclic tetrahydrooxazolo[4,5-h]-[3]benzazepin-9-ols 22 were designed as phenol bioisosteres of tetrahydro-3-benzazepine-1,7-diols. Key features of the synthesis are the introduction of the trifluoromethylsulfonyl and allyl protective groups at the heterocyclic N-atoms. Two methods were developed to convert the triflyl-protected ketone 16 into tricyclic alcohols 21 bearing various N-substituents. According to the first method, trifluoromethanesulfinate was removed by K2 CO3 . Following the selective reduction of the imino moiety of 17 with NaBH(OAc)3 afforded the aminoketone 18, which was reductively alkylated and reduced. According to the second method, both the imine and the ketone of the iminoketone 17 were reduced with NaBH4 to yield the aminoalcohol 20, which was alkylated or reductively alkylated to form tertiary amines 21f-21r. In the last step, the allyl protective group of 21 was removed with RhCl3 and HCl to obtain oxazolones 22. In receptor binding studies using [3 H]ifenprodil as radioligand ketone, 22m showed the highest GluN2B affinity (Ki = 88 nM). However, a reduced affinity toward GluN2B subunit-containing N-methyl- d-aspartate (NMDA) receptors was observed for oxazolones 22 compared to bioisosteric 3-benzazepine-1,7-diols. High selectivity of 22m for the ifenprodil binding site of GluN2B-NMDA receptors over the 1-(1-phenylcyclohexyl)piperidine binding site and σ2 receptors was observed, but only negligible selectivity over σ1 receptors. In two-electrode voltage clamp experiments, the 4-phenylbutyl derivative 22d (Ki = 422 nM) demonstrated 80% inhibition of ion flux at a concentration of 1 µM. The differences in GluN2B affinity and inhibitory activity are explained by docking studies. In conclusion, 22d is regarded as a novel scaffold of highly potent GluN1/GluN2B antagonists.

Phenol-benzoxazolone bioisosteres of GluN2B-NMDA receptor antagonists: Unexpected rearrangement during reductive alkylation with phenylcyclohexanone.

Negative allosteric modulators of N-methyl- d-aspartate receptors containing the GluN2B subunit represent promising drug candidates for the treatment of various neurological disorders including stroke, epilepsy, and Parkinson's disease. To increase the bioavailability and GluN2B affinity, the phenol of the potent benzazepine-based inhibitor, WMS-1410 (3), was replaced bioisosterically by a benzoxazolone moiety and the phenylbutyl side chain was conformationally restricted in a phenylcyclohexyl substituent. A four-step, one-pot procedure transformed the oxazolo-benzazepine 7 into the phenylcyclohexyl derivative 11. The same protocol was applied to the methylated analog 12, which unexpectedly led to ring-contracted oxazolo-isoquinolines 18. This rearrangement was explained by the additional methyl moiety in the 8-position inhibiting the formation of the planar intermediate iminium ion with phenylcyclohexanone. The allyl protective group of 11 and 18 was removed with RhCl3 and HCl to obtain the tricyclic compounds 5 and 19 without substituent at the oxazolone ring. The structures of the rearranged products 18 and 19 were elucidated by X-ray crystal structure analysis. The oxazolo-isoquinoline trans-18 with allyl moiety (Ki = 89 nM) and the oxazolo-benzazepine 5 without substituent at the oxazolone ring (Ki = 114 nM) showed GluN2B affinity in the same range as the lead compound 3. In two-electrode voltage clamp measurements, 5 displayed only weak inhibitory activity.

Effects of Axonal Demyelination, Inflammatory Cytokines and Divalent Cation Chelators on Thalamic HCN Channels and Oscillatory Bursting.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1ß; IL-6; INF-α; INF-ß) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (Ih) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of Ih (potential of half-maximal activation (V0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, Ih was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-α and IL-1ß. It was found that Ih parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1ß increased the availability of HCN channels (depolarized V0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-α mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1ß and INF-α increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines.

Beyond Hot and Spicy: TRPV Channels and their Pharmacological Modulation.

Transient receptor potential vanilloid (TRPV) channels are part of the TRP channel superfamily and named after the first identified member TRPV1, that is sensitive to the vanillylamide capsaicin. Their overall structure is similar to the structure of voltage gated potassium channels (Kv) built up as homotetramers from subunits with six transmembrane helices (S1-S6). Six TRPV channel subtypes (TRPV1-6) are known, that can be subdivided into the thermoTRPV (TRPV1-4) and the Ca2+-selective TRPV channels (TRPV5, TRPV6). Contrary to Kv channels, TRPV channels are not primary voltage gated. All six channels have distinct properties and react to several endogenous ligands as well as different gating stimuli such as heat, pH, mechanical stress, or osmotic changes. Their physiological functions are highly diverse and subtype as well as tissue specific. In many tissues they serve as sensors for different pain stimuli (heat, pressure, pH) and contribute to the homeostasis of electrolytes, the maintenance of barrier functions and the development of macrophages. Due to their fundamental role in manifold physiological and pathophysiological processes, TRPV channels are promising targets for drug development. However, drugs targeting specific TRPV channels, that are suitable for drug therapy, are rare. Moreover, selective and potent compounds for further research at TRPV channels are often lacking. In this review different aspects of the structure, the different gating stimuli, the expression pattern, the physiological and pathophysiological roles as well as the modulating mechanisms of synthetic, natural and endogenous ligands are summarized.

Virus-Host Interactions of Enteroviruses and Parvovirus B19 in Myocarditis.

Viral diseases are a major threat to modern society and the global health system. It is therefore of utter relevance to understand the way viruses affect the host as a basis to find new treatment solutions. The understanding of viral myocarditis (VMC) is incomplete and effective treatment options are lacking. This review will discuss the mechanism, effects, and treatment options of the most frequent myocarditis-causing viruses namely enteroviruses such as Coxsackievirus B3 (CVB3) and Parvovirus B19 (PVB19) on the human heart. Thereby, we focus on: 1. Viral entry: CVB3 use Coxsackievirus-Adenovirus-Receptor (CAR) and Decay Accelerating Factor (DAF) to enter cardiac myocytes while PVB19 use the receptor globoside (Gb4) to enter cardiac endothelial cells. 2. Immune system responses: The innate immune system mediated by activated cardiac toll-like receptors (TLRs) worsen inflammation in CVB3-infected mouse hearts. Different types of cells of the adaptive immune system are recruited to the site of inflammation that have either protective or adverse effects during VMC. 3. Autophagy: CVB3 evades autophagosomal degradation and misuses the autophasomal pathway for viral replication and release. 4. Viral replication sites: CVB3 promotes the formation of double membrane vesicles (DMVs), which it uses as replication sites. PVB19 uses the host cell nucleus as the replication site and uses the host cell DNA replication system. 5. Cell cycle manipulation: CVB3 attenuates the cell cycle at the G1/S phase, which promotes viral transcription and replication. PVB19 exerts cell cycle arrest in the S phase using its viral endonuclease activity. 6. Regulation of apoptosis: Enteroviruses prevent apoptosis during early stages of infection and promote cell death during later stages by using the viral proteases 2A and 3C, and viroporin 2B. PVB19 promotes apoptosis using the non-structural proteins NS1 and the 11 kDa protein. 7. Energy metabolism: Dysregulation of respiratory chain complex expression, activity and ROS production may be altered in CVB3- and PVB19-mediated myocarditis. 8. Ion channel modulation: CVB3-expression was indicated to alter calcium and potassium currents in Xenopus laevis oocytes and rodent cardiomyocytes. The phospholipase 2-like activity of PVB19 may alter several calcium, potassium and sodium channels. By understanding the general pathophysiological mechanisms of well-studied myocarditis-linked viruses, we might be provided with a guideline to handle other less-studied human viruses.

Deconstruction - Reconstruction: Analysis of the Crucial Structural Elements of GluN2B-Selective, Negative Allosteric NMDA Receptor Modulators with 3-Benzazepine Scaffold.

BACKGROUND/AIMS: The NMDA receptor plays a key role in the pathogenesis of neurodegenerative disorders including Alzheimer's and Huntington's disease, as well as depression and drug or alcohol dependence. Due to its participation in these pathologies, the development of selective modulators for this ion channel is a promising strategy for rational drug therapy. The prototypical negative allosteric modulator ifenprodil inhibits selectively GluN2B subunit containing NMDA receptors. It was conformationally restricted as 2-methyl-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol, which showed high GluN2B affinity and inhibitory activity. For a better understanding of the relevance of the functional groups and structural elements, the substituents of this 3-benzazepine were removed successively (deconstruction). Then, additional structural elements were introduced (reconstruction) with the aim to analyze, which additional modifications were tolerated by the GluN2B receptor. METHODS: The GluN2B affinity was recorded in radioligand receptor binding studies with the radioligand [3H]ifenprodil. The activity of the ligands was determined in two-electrode voltage clamp experiments using Xenopus laevis oocytes transfected with cRNA encoding the GluN1-1a and GluN2B subunits of the NMDA receptor. Docking studies showed the crucial interactions with the NMDA receptor protein. RESULTS: The deconstruction approach showed that removal of the methyl moiety and the phenolic OH moiety in 7-positon resulted in almost the same GluN2B affinity as the parent 3-benzazepine. A considerably reduced GluN2B affinity was found for the 3-benzazepine without further substituents. However, removal of one or both OH moieties led to considerably reduced NMDA receptor inhibition. Introduction of a NO2 moiety or bioisosteric replacement of the phenol by a benzoxazolone resulted in comparable GluN2B affinity, but almost complete loss of inhibitory activity. An O-atom, a carbonyl moiety or a F-atom in the tetramethylene spacer led to 6-7-fold reduced ion channel inhibition. CONCLUSION: The results reveal an uncoupling of affinity and activity for the tested 3-benzazepines. Strong inhibition of [3H]ifenprodil binding by a test compound does not necessarily translate into strong inhibition of the ion flux through the NMDA receptor associated ion channel. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine- 1,7-diol (WMS-1410) shows high GluN2B affinity and strong inhibition of the ion channel. Deconstruction by removal of one or both OH moieties reduced the inhibitory activity proving the importance of the OH groups for ion channel blockade. Reconstruction by introduction of various structural elements into the left benzene ring or into the tetramethylene spacer reduced the NMDA receptor inhibition. It can be concluded that these modifications are not able to translate binding into inhibition.

Can Unlikely Neanderthal Chloride Channel CLC-2 Gene Variants Provide Insights in Modern Human Infertility?

BACKGROUND/AIMS: Neanderthals, although well adapted to local environments, were rapidly replaced by anatomically modern humans (AMH) for unknown reasons. Genetic information on Neanderthals is limited restricting applicability of standard population genetics. METHODS: Here, we apply a novel combination of restricted genetic analyses on preselected physiological key players (ion channels), electrophysiological analyses of gene variants of unclear significance expressed in Xenopus laevis oocytes using two electrode voltage clamp and transfer of results to AMH genetics. Using genetic screening in infertile men identified a loss of CLC-2 associated with sperm deficiency. RESULTS: Increased genetic variation caused functionally impaired Neanderthals CLC-2 channels. CONCLUSION: Increased genetic variation could reflect an adaptation to different local salt supplies at the cost of reduced sperm density. Interestingly and consistent with this hypothesis, lack of CLC-2 protein in a patient associates with high blood K+ concentration and azoospermia.