Elektrophysiologische Untersuchungen zur Regulation rekombinanter Kaliumkanäle durch Metabolite des Fettsäure- und Phosphoinositidstoffwechsels

Die Familie der einwärtsgleichrichtenden (Kir) K+ Kanäle ist eine wichtige Klasse von K+ Kanälen, die an der Aufrechterhaltung des Ruhemembranpotentials, der Festlegung des Schwellenwertes für Aktionspotentiale und der K+ Homöostase maßgeblich beteiligt ist. In dieser Arbeit wurde die Regulation von Kir-Kanälen durch langkettige Fettsäure-Coenzym A Ester (LC-CoA) untersucht, deren intrazelluläre Konzentration in bestimmten Stoffwechselsituationen erhöht sein kann (z. B. bei Adipositas) und so möglicherweise die Entstehung eines Diabetes mellitus vom Typ II begünstigt. Unter Anwendung der “patch-clamp“-Technik konnte gezeigt werden, dass LC-CoA potente Inhibitoren aller getesteten Kir-Kanäle (Kir1.1, Kir2.1, Kir3.4, Kir7.1) darstellen und dass die Präsenz einer bestimmten Phosphatgruppe im Coenzym A Molekül entscheidend für die Wirkung an Kir-Kanälen ist. Des Weiteren sind die Länge der Fettsäurekette und die Potenz der Inhibition direkt korreliert. Am Kanalprotein selbst führt das Binden von LC-CoA zu strukturellen Veränderungen des Kanals, es konnten sowohl intra- als auch extrazelluläre Abschnitte als Teil einer Gating-Maschinerie identifiziert werden, die das Öffnen und Schließen des Kanals vermittelt. Die Untersuchungen implizieren eine direkte Verbindung aller Kir-Kanäle mit dem zellulären Fettsäuremetabolismus und dienen dem besseren Verständnis des komplexen Schaltverhaltens von Kir-Kanälen

pH-sensitive K⁺ channel TREK-1 is a novel target in pancreatic cancer

AbstractPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and new therapeutic targets are urgently needed. One of the hallmarks of cancer is changed pH-homeostasis and potentially pH-sensors may play an important role in cancer cell behavior. Two-pore potassium channels (K2P) are pH-regulated channels that conduct a background K+ current, which is involved in setting the plasma membrane potential (Vm). Some members of the K2P superfamily were reported as crucial players in driving tumor progression. The aim of this study was to investigate pH-regulated K+ currents in PDAC cells and determine possible effects on their pathological phenotype. Using a planar high-throughput patch-clamp system (SyncroPatch 384PE) we identified a pH-regulated K+ current in the PDAC cell line BxPC-3. The current was inhibited by extracellular acidification and intracellular alkalization. Exposure to a set of different K+ channel inhibitors, and the TREK-1 (K2P2.1)–specific activator BL1249, TREK-1 was identified as the main component of pH-regulated current. A voltage-sensor dye (VF2.1.Cl) was used to monitor effects of pH and BL1249 on Vm in more physiological conditions and TREK-1–mediated current was found as critical player in setting Vm. We assessed a possible role of TREK-1 in PDAC progression using cell proliferation and migration assays and observed similar trends with attenuated proliferation/migration rates in acidic (pH<7.0) and alkaline (pH>7.4) conditions. Notably, BL1249 inhibited both PDAC cell proliferation and migration indicating that hyperpolarization of Vm attenuates cancer cell behavior. TREK-1 may therefore be a promising novel target for PDAC therapy

State-Dependent Network Connectivity Determines Gating in a K+ Channel

YesX-ray crystallography has provided tremendous insight into the different structural states of membrane proteins and, in particular, of ion channels. However, the molecular forces that determine the thermodynamic stability of a particular state are poorly understood. Here we analyze the different X-ray structures of an inwardly rectifying potassium channel (Kir1.1) in relation to functional data we obtained for over 190 mutants in Kir1.1. This mutagenic perturbation analysis uncovered an extensive, state-dependent network of physically interacting residues that stabilizes the pre-open and open states of the channel, but fragments upon channel closure. We demonstrate that this gating network is an important structural determinant of the thermodynamic stability of these different gating states and determines the impact of individual mutations on channel function. These results have important implications for our understanding of not only K+ channel gating but also the more general nature of conformational transitions that occur in other allosteric proteins.Wellcome Trus

A novel gain-of-function mutation of Piezo1 is functionally affirmed in red blood cells by high-throughput patch clamp

No Abstract available

Structural and Thermodynamic Characterization of the Gating Pathway in a K+ Channel

YesConference abstrac

The pore structure and gating mechanism of K2P channels

K2P potassium channels are important regulators of cellular excitability. This study reveals that in contrast to most other K+ channels the primary gating mechanism in the K2P channel TREK-1 does not involve opening and closure of the cytoplasmic bundle crossing, but takes place close to or within the selectivity filter

Corrigendum to “A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm” [Toxicology and Applied Pharmacology volume 394C (2020) 114961]

© 2020 The Author(s) The authors regret that one affiliation address is mistaken in the published paper. Matthew Bridgland-Taylor's affiliation was incorrectly listed as Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Cambridge, United Kingdom. The correct affiliation is Clinical Pharmacology & Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom. The authors would like to apologise for any inconvenience caused

A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm

© 2020 Introduction: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. Methods: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. Results: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. Discussion: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment

Voltage-Dependent Gating in a “Voltage Sensor-Less” Ion Channel

An unusual mechanism of ion channel regulation generates voltage-dependent gating in the absence of a canonical voltage-sensing domain