Volume 404, Issue 4

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ), a known regulator of membrane protein trafficking. PI(3,5)P 2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P 2 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)P 2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P 2 binding sites, the known PIP 2 site PS1 and the newly identified N-terminal α–helix S0 as relevant for functional PIKfyve effects. Cd 2+ 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)P 2 to both sites.

The GluN2C subunit exists predominantly, but not exclusively in NMDA receptors within the cerebellum. Antagonists such as UBP1700 and positive allosteric modulators including PYD-106 and 3-acylamino-2-aminopropionic acid derivatives such as UA3-10 (( R )-2-amino-3-{[5-(2-bromophenyl)thiophen-2-yl]carboxamido}propionic acid) represent promising tool compounds to investigate the role of GluN2C-containing NMDA receptors in the signal transduction in the brain. However, due to its high polarity the bioavailability and CNS penetration of the amino acid UA3-10 are expected to be rather low. Herein, three ester prodrugs 12a – c of the NMDA receptor glycine site agonist UA3-10 were prepared and pharmacokinetically characterized. The esters 12a – c showed higher lipophilicity (higher log D 7.4 values) than the acid UA3-10 but almost the same binding at human serum albumin. The acid UA3-10 was rather stable upon incubation with mouse liver microsomes and NADPH, but the esters 12a – c were fast hydrolyzed to afford the acid UA3-10. Incubation with pig liver esterase and mouse serum led to rapid hydrolysis of the esters 12a – c . The isopropyl ester 12c showed a promising log D 7.4 value of 3.57 and the highest stability in the presence of pig liver esterase and mouse serum. These results demonstrate that ester prodrugs of UA3-10 can potentially afford improved bioavailability and CNS penetration.

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.

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 (logD 7.4  = 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 [ 18 F] 11 and [ 18 F] 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.

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.

It is known that the thalamus plays an important role in pathological brain conditions involved in demyelinating, inflammatory and neurodegenerative diseases such as Multiple Sclerosis (MS). Beside immune cells and cytokines, ion channels were found to be key players in neuroinflammation. MS is a prototypical example of an autoimmune disease of the central nervous system that is classified as a channelopathy where abnormal ion channel function leads to symptoms and clinical signs. Here we review the influence of the cytokine-ion channel interaction in the thalamocortical system in demyelination and inflammation.

Due to its unique features amongst ionotropic glutamate receptors, the NMDA receptor is of special interest in the physiological context but even more as a drug target. In the pathophysiology of metabolic disorders, particularly type 2 diabetes mellitus, there is evidence that NMDA receptor activation contributes to disease progression by impairing beta cell function. Consequently, channel inhibitors are suggested for treatment, but up to now there are many unanswered questions about the signaling pathways NMDA receptors are interfering with in the islets of Langerhans. In this review we give an overview about channel structure and function with special regard to the pancreatic beta cells and the regulation of insulin secretion. We sum up which signaling pathways from brain research have already been transferred to the beta cell, and what still needs to be proven. The main focus is on the relationship between an over-stimulated NMDA receptor and the production of reactive oxygen species, the amount of which is crucial for beta cell function. Finally, pilot studies using NMDA receptor blockers to protect the islet from dysfunction are reviewed and future perspectives for the use of such compounds in the context of impaired glucose homeostasis are discussed.

Cancer is characterized by uncontrolled growth, invasion, and metastasis. In addition to solid cancer cells, cancer-associated fibroblasts (CAFs) play important roles in cancer pathophysiology. They arise from “healthy” cells but get manipulated by solid cancer cells to supply them and develop a tumor microenvironment (TME) that protects the cancer cells from the immune defense. A wide variety of cell types can differentiate into CAFs, including fibroblasts, endothelial cells, and epithelial cells. Precise Ca 2+ regulation is essential for each cell including CAFs. The electrogenic Na + /Ca 2+ exchanger (NCX) is one of the ubiquitously expressed regulatory Ca 2+ transport proteins that rapidly responds to changes of the intracellular ion concentrations. Its transport function is also influenced by the membrane potential and thereby indirectly by the activity of ion channels. NCX transports Ca 2+ out of the cell (forward mode) or allows its influx (reverse mode), always in exchange for 3 Na + ions that are moved into the opposite direction. In this review, we discuss the functional roles NCX has in CAFs and how these depend on the properties of the TME. NCX activity modifies migration and leads to a reduced proliferation and apoptosis. The effect of the NCX in fibrosis is still largely unknown.

Ion channels play an important role for regulation of the exocrine and the endocrine pancreas. This review focuses on the Ca 2+ -regulated K + channel K Ca 3.1, encoded by the KCNN4 gene, which is present in both parts of the pancreas. In the islets of Langerhans, K Ca 3.1 channels are involved in the regulation of membrane potential oscillations characterizing nutrient-stimulated islet activity. Channel upregulation is induced by gluco- or lipotoxic conditions and might contribute to micro-inflammation and impaired insulin release in type 2 diabetes mellitus as well as to diabetes-associated renal and vascular complications. In the exocrine pancreas K Ca 3.1 channels are expressed in acinar and ductal cells. They are thought to play a role for anion secretion during digestion but their physiological role has not been fully elucidated yet. Pancreatic carcinoma, especially pancreatic ductal adenocarcinoma (PDAC), is associated with drastic overexpression of K Ca 3.1. For pharmacological targeting of K Ca 3.1 channels, we are discussing the possible benefits K Ca 3.1 channel inhibitors might provide in the context of diabetes mellitus and pancreatic cancer, respectively. We are also giving a perspective for the use of a fluorescently labeled derivative of the K Ca 3.1 blocker senicapoc as a tool to monitor channel distribution in pancreatic tissue. In summary, modulating K Ca 3.1 channel activity is a useful strategy for exo-and endocrine pancreatic disease but further studies are needed to evaluate its clinical suitability.

Modulation of two-pore domain potassium (K 2P ) 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.