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Aims: Atrial fibrillation (AF) is associated with tachycardia-induced cellular electrophysiology alterations which promote AF chronification and treatment resistance. Development of novel antiarrhythmic therapies is hampered by the absence of scalable experimental human models that reflect AF-associated electrical remodelling. Therefore, we aimed to assess if AF-associated remodelling of cellular electrophysiology can be simulated in human atrial-like cardiomyocytes derived from induced pluripotent stem cells in the presence of retinoic acid (iPSC-aCM), and atrial-engineered human myocardium (aEHM) under short term (24 h) and chronic (7 days) tachypacing (TP)., Methods and Results: First, 24-h electrical pacing at 3 Hz was used to investigate whether AF-associated remodelling in iPSC-aCM and aEHM would ensue. Compared to controls (24 h, 1 Hz pacing) TP-stimulated iPSC-aCM presented classical hallmarks of AF-associated remodelling: (i) decreased L-type Ca2+ current (ICa,L) and (ii) impaired activation of acetylcholine-activated inward-rectifier K+ current (IK,ACh). This resulted in action potential shortening and an absent response to the M-receptor agonist carbachol in both iPSC-aCM and aEHM subjected to TP. Accordingly, mRNA expression of the channel-subunit Kir3.4 was reduced. Selective IK,ACh blockade with tertiapin reduced basal inward-rectifier K+ current only in iPSC-aCM subjected to TP, thereby unmasking an agonist-independent constitutively active IK,ACh. To allow for long-term TP, we developed iPSC-aCM and aEHM expressing the light-gated ion-channel f-Chrimson. The same hallmarks of AF-associated remodelling were observed after optical-TP. In addition, continuous TP (7 days) led to (i) increased amplitude of inward-rectifier K+ current (IK1), (ii) hyperpolarization of the resting membrane potential, (iii) increased action potential-amplitude and upstroke velocity as well as (iv) reversibly impaired contractile function in aEHM., Conclusions: Classical hallmarks of AF-associated remodelling were mimicked through TP of iPSC-aCM and aEHM. The use of the ultrafast f-Chrimson depolarizing ion channel allowed us to model the time-dependence of AF-associated remodelling in vitro for the first time. The observation of electrical remodelling with associated reversible contractile dysfunction offers a novel platform for human-centric discovery of antiarrhythmic therapies., Competing Interests: Conflict of interest: W.H.Z. is founder and advisor of Repairon GmbH and myriamed GmbH. M.T. is advisor of Repairon GmbH and myriamed GmbH. myriamed GmbH commercializes iPSC-based cell and tissue models for drug discovery. M.Rap. is an employee of Nanion Technologies GmbH. This manuscript was handled by Consulting Editor David Eisner., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

PIEZO1 channels are mechanically activated cation channels that play a pivotal role in sensing mechanical forces in various cell types. Their dysfunction has been associated with numerous pathophysiological states, including generalized lymphatic dysplasia, varicose vein disease, and hereditary xerocytosis. Given their physiological relevance, investigating PIEZO1 is crucial for the pharmaceutical industry, which requires scalable techniques to allow for drug discovery. In this regard, several studies have used high-throughput automated patch clamp (APC) combined with Yoda1, a specific gating modifier of PIEZO1 channels, to explore the function and properties of PIEZO1 in heterologous expression systems, as well as in primary cells. However, a combination of solely mechanical stimulation (M-Stim) and high-throughput APC has not yet been available for the study of PIEZO1 channels. Here, we show that optimization of pipetting parameters of the SyncroPatch 384 coupled with multihole NPC-384 chips enables M-Stim of PIEZO1 channels in high-throughput electrophysiology. We used this approach to explore differences between the response of mouse and human PIEZO1 channels to mechanical and/or chemical stimuli. Our results suggest that applying solutions on top of the cells at elevated pipetting flows is crucial for activating PIEZO1 channels by M-Stim on the SyncroPatch 384. The possibility of comparing and combining mechanical and chemical stimulation in a high-throughput patch clamp assay facilitates investigations on PIEZO1 channels and thereby provides an important experimental tool for drug development., (© 2023 Murciano et al.)

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used for personalised medicine and preclinical cardiotoxicity testing. Reports on hiPSC-CM commonly describe heterogenous functional readouts and underdeveloped or immature phenotypical properties. Cost-effective, fully defined monolayer culture is approaching mainstream adoption; however, the optimal age at which to utilise hiPSC-CM is unknown. In this study, we identify, track and model the dynamic developmental behaviour of key ionic currents and Ca 2+ -handling properties in hiPSC-CM over long-term culture (30-80 days). hiPSC-CMs > 50 days post differentiation show significantly larger I Ca,L density along with an increased I Ca,L -triggered Ca 2+ -transient. I Na and I K1 densities significantly increase in late-stage cells, contributing to increased upstroke velocity and reduced action potential duration, respectively. Importantly, our in silico model of hiPSC-CM electrophysiological age dependence confirmed I K1 as the key ionic determinant of action potential shortening in older cells. We have made this model available through an open source software interface that easily allows users to simulate hiPSC-CM electrophysiology and Ca 2+ -handling and select the appropriate age range for their parameter of interest. This tool, together with the insights from our comprehensive experimental characterisation, could be useful in future optimisation of the culture-to-characterisation pipeline in the field of hiPSC-CM research., (© 2023. The Author(s).)

Despite the identification of the high-incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other 2 members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout, and expression studies in an erythroblast cell line. We report the molecular bases of 5 Er blood group antigens: the recognized Era, Erb, and Er3 antigens and 2 novel high-incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn. Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low-abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Crucial conventional patch-clamp approaches to investigate cellular electrophysiology suffer from low-throughput and require considerable experimenter expertise. Automated patch-clamp (APC) approaches are more experimenter independent and offer high-throughput, but by design are predominantly limited to assays containing small, homogenous cells. In order to enable high-throughput APC assays on larger cells such as native cardiomyocytes isolated from mammalian hearts, we employed a fixed-well APC plate format. A broad range of detailed electrophysiological parameters including action potential, L-type calcium current and basal inward rectifier current were reliably acquired from isolated swine atrial and ventricular cardiomyocytes using APC. Effective pharmacological modulation also indicated that this technique is applicable for drug screening using native cardiomyocyte material. Furthermore, sequential acquisition of multiple parameters from a single cell was successful in a high throughput format, substantially increasing data richness and quantity per experimental run. When appropriately expanded, these protocols will provide a foundation for effective mechanistic and phenotyping studies of human cardiac electrophysiology. Utilizing scarce biopsy samples, regular high throughput characterization of primary cardiomyocytes using APC will facilitate drug development initiatives and personalized treatment strategies for a multitude of cardiac diseases., (© 2022. The Author(s).)

Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (Na V ) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as Na V 1.5, Na V 1.7, and K Ca 3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions., Competing Interests: MR, AO, SS, IR-W, TG, NB, MGR, TS, AR, SF, and NF were employed by Nanion Technologies, the manufacturers of the SyncroPatch 384 used to compile this manuscript. NF was a shareholder in Nanion Technologies. EH was employed by BioPharmaceuticals R&D, AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rapedius, Obergrussberger, Humphries, Scholz, Rinke-Weiss, Goetze, Brinkwirth, Rotordam, Strassmaier, Randolph, Friis, Liutkute, Seibertz, Voigt and Fertig.)

Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with gigaohm seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPR. Additionally, we tested the effect of 36 arthropod venoms on Na V 1.9 using a single 384-well plate on the SyncroPatch 384. As an example for mutant screening, a range of acid-sensing ion channel variants were tested and the success rate increased through fluorescence-activated cell sorting (FACS) prior to APC experiments. Gigaohm seal data quality makes the 384-format accessible to recording of primary and stem cell-derived cells on the SyncroPatch 384. We show recordings in voltage and current clamp modes of stem cell-derived cardiomyocytes. In addition, the option of intracellular solution exchange enabled investigations into the effects of intracellular Ca 2+ and cAMP on TRPC5 and HCN2 currents, respectively. Together, these data highlight the broad applicability and versatility of APC platforms and also outlines some limitations of the approach. KEY POINTS: High throughput automated patch clamp (APC) can be used for a variety of applications involving ion channels. Lower false positive rates were achieved using automated patch clamp versus a fluorometric imaging plate reader (FLIPR) in a high throughput compound screen against NMDA receptors.  Genetic variants and mutations can be screened on a single 384-well plate to reduce variability of experimental parameters. Intracellular solution can be perfused to investigate effects of ions and second messenger systems without the need for excised patches. Primary cells and stem cell-derived cells can be used on high throughput APC with reasonable success rates for cell capture, voltage clamp measurements and action potential recordings in current clamp mode., (© 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Introduction: For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including Na V 1.5 and Ca V 1.2. We identified different parameters that might affect IC 50 s of compounds on Na V 1.5 peak and late currents recorded using automated patch clamp (APC) and suggest outlines for best practices., Methods: APC instruments SyncroPatch 384 and Patchliner were used to record Na V 1.5 peak and late current. Up to 24 CiPA compounds were used to investigate effects of voltage protocol, holding potential (-80 mV or - 95 mV) and temperature (23 ± 1 °C or 36 ± 1 °C) on IC 50 values on hNa V 1.5 overexpressed in HEK or CHO cells either as frozen cells or running cultures., Results: The IC 50 s of 18 compounds on the Na V 1.5 peak current recorded on the SyncroPatch 384 using the CiPA step-ramp protocol correlated well with the literature. The use of frozen or cultured cells did not affect IC 50 s but voltage protocol and holding potential did cause differences in IC 50 values. Temperature can affect V half of inactivation and also compound potency. A compound incubation time of 5-6 min was sufficient for most compounds, however slow acting compounds such as terfenadine required longer to reach maximum effect., Discussion: We conclude that holding potential, voltage protocol and temperature can affect IC 50 values and recommend the use of the CiPA step-ramp protocol at physiological temperature to record Na V 1.5 peak and late currents for cardiac safety. Further recommendations include: a minimum compound incubation time of 5 min, a replicate number of 4 and the use of positive and negative controls for reliable IC 50 s., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet., Methods: The proposed implementation strategies were applied to the CiPA model by performing a sensitivity analysis to identify a subset of calibration drugs that were most critical in determining the classification thresholds for proarrhythmia risk prediction., Results: The selected calibration drugs were able to recapitulate classification thresholds close to those calculated from the full list of CiPA drugs. Using an illustrative dataset it was shown that a new lab could use these calibration drugs to establish its own classification thresholds (lab-specific calibration), and verify that the model prediction accuracy in the new lab is comparable to that in the original lab where the model was developed (lab-specific validation)., Discussion: This work used the CiPA model as an example to illustrate how to adopt the proposed model implementation strategies to select calibration drugs and perform lab-specific calibration and lab-specific validation. Generic in nature, these strategies could be generally applied to different proarrhythmia risk prediction models using various experimental systems under the new paradigm., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest., (Published by Elsevier Inc.)

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., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Automated patch clamp (APC) devices have become commonplace in many industrial and academic labs. Their ease-of-use and flexibility have ensured that users can perform routine screening experiments and complex kinetic experiments on the same device without the need for months of training and experience. APC devices are being developed to increase throughput and flexibility. Areas covered: Experimental options such as temperature control, internal solution exchange and current clamp have been available on some APC devices for some time, and are being introduced on other devices. A comprehensive review of the literature pertaining to these features for the Patchliner, QPatch and Qube and data for these features for the SyncroPatch 384/768PE, is given. In addition, novel features such as dynamic clamp on the Patchliner and light stimulation of action potentials using channelrhodosin-2 is discussed. Expert opinion: APC devices will continue to play an important role in drug discovery. The instruments will be continually developed to meet the needs of HTS laboratories and for basic research. The use of stem cells and recordings in current clamp mode will increase, as will the development of complex add-ons such as dynamic clamp and optical stimulation on high throughput devices.

We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration-response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNa V 1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNa V 1.7 and hERG is described. Fast external solution exchange rates of <50 ms are demonstrated using Kir2.1. Short exposure times are achieved by stacking the external solutions inside the pipette of the robot to minimize exposure of the ligand on the receptor. This ensures that ligand-gated ion channels, for example, GABA and glutamate described in this report, can be reproducibly recorded. Stem cell-derived cardiomyocytes have also been used with success rates of 52% for cells that have a seal resistance of >200 MΩ, and recordings of voltage-gated Na + and Ca 2+ are shown., (© 2015 Society for Laboratory Automation and Screening.)

Pancreatic 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., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

X-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., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a 'catalytic' effect upon the channel gating kinetics.

Most known small-molecule inhibitors of voltage-gated ion channels have poor subtype specificity because they interact with a highly conserved binding site in the central cavity. Using alanine-scanning mutagenesis, electrophysiological recordings and molecular modeling, we have identified a new drug-binding site in Kv1.x channels. We report that Psora-4 can discriminate between related Kv channel subtypes because, in addition to binding the central pore cavity, it binds a second, less conserved site located in side pockets formed by the backsides of S5 and S6, the S4-S5 linker, part of the voltage sensor and the pore helix. Simultaneous drug occupation of both binding sites results in an extremely stable nonconducting state that confers high affinity, cooperativity, use-dependence and selectivity to Psora-4 inhibition of Kv1.x channels. This new mechanism of inhibition represents a molecular basis for the development of a new class of allosteric and selective voltage-gated channel inhibitors.

We previously reported that TREK-1 gating by internal pH and pressure occurs close to or within the selectivity filter. These conclusions were based upon kinetic measurements of high-affinity block by quaternary ammonium (QA) ions that appeared to exhibit state-independent accessibility to their binding site within the pore. Intriguingly, recent crystal structures of two related K2P potassium channels were also both found to be open at the helix bundle crossing. However, this did not exclude the possibility of gating at the bundle crossing and it was suggested that side-fenestrations within these structures might allow state-independent access of QA ions to their binding site. In this addendum to our original study we demonstrate that even hydrophobic QA ions do not access the TREK-1 pore via these fenestrations. Furthermore, by using a chemically reactive QA ion immobilized within the pore via covalent cysteine modification we provide additional evidence that the QA binding site remains accessible to the cytoplasm in the closed state. These results support models of K2P channel gating which occur close to or within the selectivity filter and do not involve closure at the helix bundle crossing.

Mutations of the KCNJ10 (Kir4.1) K(+) channel underlie autosomal recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy (EAST) syndrome. We investigated the localization of KCNJ10 and the homologous KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral membrane of mouse distal convoluted tubules, connecting tubules, and cortical collecting ducts. In the human kidney, KCNJ10 staining was additionally observed in the basolateral membrane of the cortical thick ascending limb of Henle's loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced basal infoldings in this nephron segment, which likely reflects the morphological consequences of the impaired salt reabsorption capacity. When expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q caused a marked impairment of channel function. R199X showed complete loss of function. Single-channel analysis revealed a strongly reduced mean open time. Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in native renal KCNJ10/KCNJ16 heteromers. The decrease in the current of R65P and R175Q was mainly caused by a remarkable shift of pH sensitivity to the alkaline range. In summary, EAST mutations of KCNJ10 lead to impaired channel function and structural changes in distal convoluted tubules. Intriguingly, the metabolic alkalosis present in patients carrying the R65P mutation possibly improves residual function of KCNJ10, which shows higher activity at alkaline pH.

The time course of inactivation of voltage-activated potassium (Kv) channels is an important determinant of the firing rate of neurons. In many Kv channels highly unsaturated lipids as arachidonic acid, docosahexaenoic acid and anandamide can induce fast inactivation. We found that these lipids interact with hydrophobic residues lining the inner cavity of the pore. We analysed the effects of these lipids on Kv1.1 current kinetics and their competition with intracellular tetraethylammonium and Kvbeta subunits. Our data suggest that inactivation most likely represents occlusion of the permeation pathway, similar to drugs that produce 'open-channel block'. Open-channel block by drugs and lipids was strongly reduced in Kv1.1 channels whose amino acid sequence was altered by RNA editing in the pore cavity, and in Kv1.x heteromeric channels containing edited Kv1.1 subunits. We show that differential editing of Kv1.1 channels in different regions of the brain can profoundly alter the pharmacology of Kv1.x channels. Our findings provide a mechanistic understanding of lipid-induced inactivation and establish RNA editing as a mechanism to induce drug and lipid resistance in Kv channels.

Background: 5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5HD + CoA + ATP --> 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K(ATP) (sarcK(ATP)) channel activity., Methods: Single sarcK(ATP) channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK(ATP) channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A., Results: 5-HD inhibited (IC(50) approximately 30 microM) K(ATP) channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 microM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 microM coenzyme A (CoA) inhibited sarcK(ATP) channels. Using giant inside-out patches, which are weakly sensitive to "contaminating" CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK(ATP) channel activation by either metabolic inhibition or rilmakalim., General Significance: SarcK(ATP) channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at "directly" inhibiting sarcK(ATP) and mitoK(ATP) channels. However, in intact cells, 5-HD fails to inhibit sarcK(ATP) channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

The voltage sensitivity of voltage-gated cation channels is primarily attributed to conformational changes of a four transmembrane segment voltage-sensing domain, conserved across many levels of biological complexity. We have identified a remarkable point mutation that confers significant voltage dependence to Kir6.2, a ligand-gated channel that lacks any canonical voltage-sensing domain. Similar to voltage-dependent Kv channels, the Kir6.2[L157E] mutant exhibits time-dependent activation upon membrane depolarization, resulting in an outwardly rectifying current-voltage relationship. This voltage dependence is convergent with the intrinsic ligand-dependent gating mechanisms of Kir6.2, since increasing the membrane PIP2 content saturates Po and eliminates voltage dependence, whereas voltage activation is more dramatic when channel Po is reduced by application of ATP or poly-lysine. These experiments thus demonstrate an inherent voltage dependence of gating in a "ligand-gated" K+ channel, and thereby provide a new view of voltage-dependent gating mechanisms in ion channels. Most interestingly, the voltage- and ligand-dependent gating of Kir6.2[L157E] is highly sensitive to intracellular [K+], indicating an interaction between ion permeation and gating. While these two key features of channel function are classically dealt with separately, the results provide a framework for understanding their interaction, which is likely to be a general, if latent, feature of the superfamily of cation channels., Competing Interests: The authors have declared that no competing interests exist.

Inhibition by intracellular H(+) (pH gating) and activation by phosphoinositides such as PIP(2) (PIP(2)-gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP(2) gating and pH gating are controlled by an intra-subunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP(2) and pH gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP2 gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP(2) affinities. This indicates that Kir channel PIP(2) affinity has little impact on either the PIP(2) or pH gating kinetics.

Specific stimuli such as intracellular H+ and phosphoinositides (e.g., PIP2) gate inwardly rectifying potassium (Kir) channels by controlling the reversible transition between the closed and open states. This gating mechanism underlies many aspects of Kir channel physiology and pathophysiology; however, its structural basis is not well understood. Here, we demonstrate that H+ and PIP2 use a conserved gating mechanism defined by similar structural changes in the transmembrane (TM) helices and the selectivity filter. Our data support a model in which the gating motion of the TM helices is controlled by an intrasubunit hydrogen bond between TM1 and TM2 at the helix-bundle crossing, and we show that this defines a common gating motif in the Kir channel superfamily. Furthermore, we show that this proposed H-bonding interaction determines Kir channel pH sensitivity, pH and PIP2 gating kinetics, as well as a K+-dependent inactivation process at the selectivity filter and therefore many of the key regulatory mechanisms of Kir channel physiology.

Long-chain fatty acids acyl coenzyme A esters (LC-CoA) are obligate intermediates of fatty acid metabolism and have been shown to activate K(ATP) channels but to inhibit most other Kir channels (e.g. Kir2.1) by direct channel binding. The activation of K(ATP) channels by elevated levels of LC-CoA may be involved in the pathophysiology of type 2 diabetes, the hypothalamic sensing of circulating fatty acids and the regulation of cardiac K(ATP) channels. However, LC-CoA are effectively buffered in the cytoplasm and it is currently not clear whether their free concentration can reach levels sufficient to affect Kir channels in vivo. Here, we report that extracellular oleic acid complexed with albumin at an unbound concentration of 81 +/- 1 nm strongly activated K(ATP) channels and inhibited Kir2.1 channels in Chinese hamster ovary (CHO) cells as well as endogenous Kir currents in human embryonic kidney (HEK293) cells. These effects were only seen in the presence of a high concentration of glucose (25 mm), a condition known to promote the accumulation of LC-CoA by inhibiting their mitochondrial uptake via carnitine-palmitoyl-transferase-1 (CPT1). Accordingly, pharmacological inhibition of CPT1 by etomoxir restored the effects of oleic acid under low glucose conditions. Finally, triacsin C, an inhibitor of the acyl-CoA synthetase, which is necessary for LC-CoA formation, abolished the effects of extracellular oleic acid on the various Kir channels. These results establish the direct regulation of Kir channels by the cytoplasmic accumulation of LC-CoA, which might be of physiological and pathophysiological relevance in a variety of tissues.

The pH-sensitive renal potassium channel Kir1.1 is important for K+ homeostasis. Disruption of the pH-sensing mechanism causes type II Bartter syndrome. The pH sensor is thought to be an anomalously titrated lysine residue (K80) that interacts with two arginine residues as part of an 'RKR triad'. We show that a Kir1.1 orthologue from Fugu rubripes lacks this lysine and yet is still highly pH sensitive, indicating that K80 is not the H+ sensor. Instead, K80 functionally interacts with A177 on transmembrane domain 2 at the 'helix-bundle crossing' and controls the ability of pH-dependent conformational changes to induce pore closure. Although not required for pH inhibition, K80 is indispensable for the coupling of pH gating to the extracellular K+ concentration, explaining its conservation in most Kir1.1 orthologues. Furthermore, we demonstrate that instead of interacting with K80, the RKR arginine residues form highly conserved inter- and intra-subunit interactions that are important for Kir channel gating and influence pH sensitivity indirectly.

Long chain fatty acid esters of coenzyme A (LC-CoA) are potent activators of ATP-sensitive (K(ATP)) channels, and elevated levels have been implicated in the pathophysiology of type 2 diabetes. This stimulatory effect is thought to involve a mechanism similar to phosphatidylinositol 4,5-bisphosphate (PIP2), which activates all known inwardly rectifying potassium (Kir) channels. However, the effect of LC-CoA on other Kir channels has not been well characterized. In this study, we show that in contrast to their stimulatory effect on K(ATP) channels, LC-CoA (e.g. oleoyl-CoA) potently and reversibly inhibits all other Kir channels tested (Kir1.1, Kir2.1, Kir3.4, Kir7.1). We also demonstrate that the inhibitory potency of the LC-CoA increases with the chain length of the fatty acid chain, while both its activatory and inhibitory effects critically depend on the presence of the 3'-ribose phosphate on the CoA group. Biochemical studies also demonstrate that PIP2 and LC-CoA bind with similar affinity to the C-terminal domains of Kir2.1 and Kir6.2 and that PIP2 binding can be competitively antagonized by LC-CoA, suggesting that the mechanism of LC-CoA inhibition involves displacement of PIP2. Furthermore, we demonstrate that in contrast to its stimulatory effect on K(ATP) channels, phosphatidylinositol 3,4-bisphosphate has an inhibitory effect on Kir1.1 and Kir2.1. These results demonstrate a bi-directional modulation of Kir channel activity by LC-CoA and phosphoinositides and suggest that changes in fatty acid metabolism (e.g. LC-CoA production) could have profound and widespread effects on cellular electrical activity.

The aqueous penetration of the commercial preparations of the fluoroquinolone antibiotics ofloxacin, ciprofloxacin, levofloxacin, and gatifloxacin were compared following topical dosing in a rabbit model. Levofloxacin achieved the highest aqueous concentrations, with a mean aqueous level of 4.8014 microcg/mL (p = 0.002, p = 0.00002, p = 0.015.) Ofloxacin (2.5136 microcg/mL) and gatifloxacin (2.4817 microcg/mL) achieved statistically equal aqueous concentrations (p = 0.479). Ciprofloxacin reached the lowest levels in the aqueous humor (0.9616 microcg/mL, p = 0.00002, 0.00004, 0.008). Gatifloxacin alone achieved concentrations in excess of the MIC90s of gram-positive pathogens of concern.

Phosphatidylinositol phosphates (PIPs, e.g. PIP2) and long-chain acyl-CoA esters (e.g. oleoyl-CoA) are potent activators of KATP channels that are thought to link KATP channel activity to the cellular metabolism of PIPs and fatty acids. Here we show that the two types of lipid act by the same mechanism: oleoyl-CoA potently reduced the ATP sensitivity of cardiac (Kir6.2/SUR2A) and pancreatic (Kir6.2/SUR1) KATP channels in a way very similar to PIP2. Mutations (R54Q, R176A) in the C- and N-terminus of Kir6.2 that greatly reduced the PIP2 modulation of ATP sensitivity likewise reduced the modulation by oleoyl-CoA, indicating that the two lipids interact with the same site. Polyvalent cations reduced the effect of oleoyl-CoA and PIP2 on the ATP sensitivity with similar potency suggesting that electrostatic interactions are of similar importance. However, experiments with differently charged inhibitory adenosine phosphates (ATP4-, ADP3- and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP2-)) and diadenosine tetraphosphate (Ap4A5-) ruled out a mechanism where oleoyl-CoA or PIP2 attenuate ATP inhibition by reducing ATP binding through electrostatic repulsion. Surprisingly, CoA (the head group of oleoyl-CoA) did not activate but inhibited KATP channels (IC50 = 265 +/- 33 muM). We provide evidence that CoA and diadenosine polyphosphates (e.g. Ap4A) are ligands of the inhibitory ATP-binding site on Kir6.2.

Purpose: The major goal of this study was to compare the relative utility of the Hanson Microette and the Van Kel apparatus, two fully automated devices, as in vitro release tests (IVRT) for semisolids. We attempted to develop methodology that can be used to discriminate formulation changes, and to evaluate the precision, reproducibility and technical complexity of each test apparatus., Methods: We chose the sunscreen Eusolex 232 (2-Phenylbenzimidazole-5-sulfonic acid) as a model compound, which was incorporated into an emulsion formulation prepared in our laboratory. Test conditions for the two IVRT were made as nearly identical as possible, in order to obtain an accurate comparison., Results: The formulations were tested and found to be physically stable throughout the entire study. Diffusion coefficients were apparatus-dependent but were independent of the drug concentration in the formulations. The IVRT data were plotted as amount released (microg/cm2) vs. square root of time (s(0.5)) and a linear relationship was obtained in each case. Both methods produced similar results and were able to detect changes in drug loading in the formulations., Conclusions: The linear relationship between the amount released and the square root of time indicates a diffusion-controlled release of drug. Both apparatuses proved to be suitable as tests for formulation " sameness" according to the FDA's SUPAC-SS guidelines, during level 3 changes. However, each apparatus produced a different release profile for the drug. The choice of apparatus will depend upon a number of considerations.