Activation of hTREK-1 by polyunsaturated fatty acids involves direct interaction.

TREK-1 is a mechanosensitive channel activated by polyunsaturated fatty acids (PUFAs). Its activation is supposed to be linked to changes in membrane tension following PUFAs insertion. Here, we compared the effect of 11 fatty acids and ML402 on TREK-1 channel activation using the whole cell and the inside-out configurations of the patch-clamp technique. Firstly, TREK-1 activation by PUFAs is variable and related to the variable constitutive activity of TREK-1. We observed no correlation between TREK-1 activation and acyl chain length or number of double bonds suggesting that the bilayer-couple hypothesis cannot explain by itself the activation of TREK-1 by PUFAs. The membrane fluidity measurement is not modified by PUFAs at 10 µM. The spectral shift analysis in TREK-1-enriched microsomes indicates a KD,TREK1 at 44 µM of C22:6 n-3. PUFAs display the same activation and reversible kinetics than the direct activator ML402 and activate TREK-1 in both whole-cell and inside-out configurations of patch-clamp suggesting that the binding site of PUFAs is accessible from both sides of the membrane, as for ML402. Finally, we proposed a two steps mechanism: first, insertion into the membrane, with no fluidity or curvature modifications at 10 µM, and then interaction with TREK-1 channel to open it.

Structured connectivity in the output of the cerebellar cortex.

The spatial organization of a neuronal circuit is critically important for its function since the location of neurons is often associated with function. In the cerebellum, the major output of the cerebellar cortex are synapses made from Purkinje cells onto neurons in the cerebellar nuclei, yet little has been known about the spatial organization of these synapses. We explored this question using whole-cell electrophysiology and optogenetics in acute sagittal cerebellar slices to produce spatial connectivity maps of cerebellar cortical output in mice. We observed non-random connectivity where Purkinje cell inputs clustered in cerebellar transverse zones: while many nuclear neurons received inputs from a single zone, several multi-zonal connectivity motifs were also observed. Single neurons receiving input from all four zones were overrepresented in our data. These findings reveal that the output of the cerebellar cortex is spatially structured and represents a locus for multimodal integration in the cerebellum.

Electrophysiological Characterization of Inducible Pluripotent Stem Cell-Derived Human ß-Like Cells and an SLC30A8 Disease Model.

Inducible pluripotent stem cell-derived human ß-like cells (BLCs) hold promise for both therapy and disease modeling, but their generation remains challenging and their functional analyses beyond transcriptomic and morphological assessments remain limited. Here, we validate an approach using multicellular and single-cell electrophysiological tools to evaluate function of BLCs from pioneer protocols that can be easily adapted to more differentiated BLCs. The multi-electrode arrays (MEAs) measuring the extracellular electrical activity revealed that BLCs, like primary ß-cells, are electrically coupled and produce slow potential (SP) signals that are closely linked to insulin secretion. We also used high-resolution single-cell patch clamp measurements to capture the exocytotic properties, and characterize voltage-gated sodium and calcium currents, and found that they were comparable with those in primary ß- and EndoC-ßH1 cells. The KATP channel conductance is greater than in human primary ß-cells, which may account for the limited glucose responsiveness observed with MEA. We used MEAs to study the impact of the type 2 diabetes-protective SLC30A8 allele (p.Lys34Serfs50*) and found that BLCs with this allele have stronger electrical coupling activity. Our data suggest that BLCs can be used to evaluate the functional impact of genetic variants on ß-cell function and coupling.

Differential potassium channel inhibitory activities of a novel thermostable degradation peptide BmKcug1a-P1 from scorpion medicinal material and its N-terminal truncated/restored peptides.

Thermally stable full-length scorpion toxin peptides and partially degraded peptides with complete disulfide bond pairing are valuable natural peptide resources in traditional Chinese scorpion medicinal material. However, their pharmacological activities are largely unknown. This study discovered BmKcug1a-P1, a novel N-terminal degraded peptide, in this medicinal material. BmKcug1a-P1 inhibited hKv1.2 and hKv1.3 potassium channels with IC50 values of 2.12 ± 0.27 µM and 1.54 ± 0.28 µM, respectively. To investigate the influence of N-terminal amino acid loss on the potassium channel inhibiting activities, three analogs (i.e., full-length BmKcug1a, BmKcug1a-P1-D2 and BmKcug1a-P1-D4) of BmKcug1a-P1 were prepared, and their potassium channel inhibiting activities on hKv1.3 channel were verified by whole-cell patch clamp technique. Interestingly, the potassium channel inhibiting activity of full-length BmKcug1a on the hKv1.3 channel was significantly improved compared to its N-terminal degraded form (BmKcug1a-P1), while the activities of two truncated analogs (i.e., BmKcug1a-P1-D2 and BmKcug1a-P1-D4) were similar to that of BmKcug1a-P1. Extensive alanine-scanning experiments identified the bonding interface (including two key functional residues, Asn30 and Arg34) of BmKcug1a-P1. Structural and functional dissection further elucidated whether N-terminal residues of the peptide are located at the bonding interface is important in determining whether the N-terminus significantly influences the potassium channel inhibiting activity of the peptide. Altogether, this research identified a novel N-terminal degraded active peptide, BmKcug1a-P1, from traditional Chinese scorpion medicinal material and elucidated how the N-terminus of peptides influences their potassium channel inhibiting activity, contributing to the functional identification and molecular truncation optimization of full-length and degraded peptides from traditional Chinese scorpion medicinal material Buthus martensii Karsch.

Outward potassium current in neurons of aestivated land snail Achatina fulica.

Aestivation and hibernation represent distinct forms of animal quiescence, characterized by physiological changes, including ion composition. Intracellular ion flows play a pivotal role in eliciting alterations in membrane potential and facilitating cellular communication, while outward K+ currents aid in the restitution and upkeep of the resting membrane potential. This study explores the relationship between inward and outward currents during aestivation in Achatina fulica snails. Specimens were collected near MSUBIT University in Shenzhen and divided into two groups. The first group was kept on a lattice diet, while the second one consisted of aestivating individuals, that were deprived of food and water until a cork-like structure sealed their shells. Recording of current from isolated neurons were conducted using the single-electrode voltage clamp mode with an AxoPatch 200B amplifier. Electrophysiological recordings on pedal ganglia neurons revealed significant differences in the inactivation processes of the Ia and Ikdr components. Alterations in the Ikdr component may inhibit pacemaker activity in pedal ganglion neurons, potentially contributing to locomotion cessation in aestivated animals. The KS current remains unaffected during aestivation. Changes in slow K+ current components could disrupt the resting membrane potential, possibly leading to cell depolarization and influx of Ca2+ and Na+ ions, impacting cell homeostasis. Thus, maintaining the constancy of outward K+ current is essential for cell stability.

Clustered synapses develop in distinct dendritic domains in visual cortex before eye opening.

Synaptic inputs to cortical neurons are highly structured in adult sensory systems, such that neighboring synapses along dendrites are activated by similar stimuli. This organization of synaptic inputs, called synaptic clustering, is required for high-fidelity signal processing, and clustered synapses can already be observed before eye opening. However, how clustered inputs emerge during development is unknown. Here, we employed concurrent in vivo whole-cell patch-clamp and dendritic calcium imaging to map spontaneous synaptic inputs to dendrites of layer 2/3 neurons in the mouse primary visual cortex during the second postnatal week until eye opening. We found that the number of functional synapses and the frequency of transmission events increase several fold during this developmental period. At the beginning of the second postnatal week, synapses assemble specifically in confined dendritic segments, whereas other segments are devoid of synapses. By the end of the second postnatal week, just before eye opening, dendrites are almost entirely covered by domains of co-active synapses. Finally, co-activity with their neighbor synapses correlates with synaptic stabilization and potentiation. Thus, clustered synapses form in distinct functional domains presumably to equip dendrites with computational modules for high-capacity sensory processing when the eyes open.

In situ Patch-seq analysis of microglia reveals a lack of stress genes as found in FACS-isolated microglia.

We applied the patch-seq technique to harvest transcripts from individual microglial cells from cortex, hippocampus and corpus callosum of acute brain slices from adult mice. After recording membrane currents with the patch-clamp technique, the cytoplasm was collected via the pipette and underwent adapted SMART-seq2 preparation with subsequent sequencing. On average, 4138 genes were detected in 113 cells from hippocampus, corpus callosum and cortex, including microglia markers such as Tmem119, P2ry12 and Siglec-H. Comparing our dataset to previously published single cell mRNA sequencing data from FACS-isolated microglia indicated that two clusters of cells were absent in our patch-seq dataset. Pathway analysis of marker genes in FACS-specific clusters revealed association with microglial activation and stress response. This indicates that under normal conditions microglia in situ lack transcripts associated with a stress-response, and that the microglia-isolation procedure by mechanical dissociation and FACS triggers the expression of genes related to activation and stress.

Automated Patch Clamp Recordings of GPCR-Gated Ion Channels: Targeting the MC4-R/Kir7.1 Potassium Channel Complex.

Automated patch clamp recording is a valuable technique in drug discovery and the study of ion channels. It allows for the precise measurement and manipulation of channel currents, providing insights into their function and modulation by drugs or other compounds. The melanocortin 4 receptor (MC4-R) is a G protein-coupled receptor (GPCR) crucial to appetite regulation, energy balance, and body weight. MC4-R signaling is complex and involves interactions with other receptors and neuropeptides in the appetite-regulating circuitry. MC4-Rs, like other GPCRs, are known to modulate ion channels such as Kir7.1, an inward rectifier potassium channel, in response to ligand binding. This modulation is critical for controlling ion flow across the cell membrane, which can influence membrane potential, excitability, and neurotransmission. The MC4-R is the target for the anti-obesity drug Imcivree. However, this drug is known to lack optimal potency and also has side effects. Using high-throughput techniques for studying the MC4-R/Kir7.1 complex allows researchers to rapidly screen many compounds or conditions, aiding the development of drugs that target this system. Additionally, automated patch clamp recording of this receptor-channel complex and its ligands can provide valuable functional and pharmacological insights supporting the development of novel therapeutic strategies. This approach can be generalized to other GPCR-gated ion channel functional complexes, potentially accelerating the pace of research in different fields with the promise to uncover previously unknown aspects of receptor-ion channel interactions.

Glycine-induced activation of GPR158 increases the intrinsic excitability of medium spiny neurons in the nucleus accumbens.

It has been recently established that GPR158, a class C orphan G protein-coupled receptor, serves as a metabotropic glycine receptor. GPR158 is highly expressed in the nucleus accumbens (NAc), a major input structure of the basal ganglia that integrates information from cortical and subcortical structures to mediate goal-directed behaviors. However, whether glycine modulates neuronal activity in the NAc through GPR158 activation has not been investigated yet. Using whole-cell patch-clamp recordings, we found that glycine-dependent activation of GPR158 increased the firing rate of NAc medium spiny neurons (MSNs) while it failed to significantly affect the excitability of cholinergic interneurons (CIN). In MSNs GPR158 activation reduced the latency to fire, increased the action potential half-width, and reduced action potential afterhyperpolarization, effects that are all consistent with negative modulation of potassium M-currents, that in the central nervous system are mainly carried out by Kv7/KCNQ-channels. Indeed, we found that the GPR158-induced increase in MSN excitability was associated with decreased M-current amplitude, and selective pharmacological inhibition of the M-current mimicked and occluded the effects of GPR158 activation. In addition, when the protein kinase A (PKA) or extracellular signal-regulated kinase (ERK) signaling was pharmacologically blocked, modulation of MSN excitability by GPR158 activation was suppressed. Moreover, GPR158 activation increased the phosphorylation of ERK and Kv7.2 serine residues. Collectively, our findings suggest that GPR158/PKA/ERK signaling controls MSN excitability via Kv7.2 modulation. Glycine-dependent activation of GPR158 may significantly affect MSN firing in vivo, thus potentially mediating specific aspects of goal-induced behaviors.

PIP2 modulates TRPC3 activity via TRP helix and S4-S5 linker.

The transient receptor potential canonical type 3 (TRPC3) channel plays a pivotal role in regulating neuronal excitability in the brain via its constitutive activity. The channel is intricately regulated by lipids and has previously been demonstrated to be positively modulated by PIP2. Using molecular dynamics simulations and patch clamp techniques, we reveal that PIP2 predominantly interacts with TRPC3 at the L3 lipid binding site, located at the intersection of pre-S1 and S1 helices. We demonstrate that PIP2 sensing involves a multistep mechanism that propagates from L3 to the pore domain via a salt bridge between the TRP helix and S4-S5 linker. Notably, we find that both stimulated and constitutive TRPC3 activity require PIP2. These structural insights into the function of TRPC3 are invaluable for understanding the role of the TRPC subfamily in health and disease, in particular for cardiovascular diseases, in which TRPC3 channels play a major role.

Cortical nitric oxide required for presynaptic long-term potentiation in the insular cortex.

Nitric oxide (NO) is a key diffusible messenger in the mammalian brain. It has been proposed that NO may diffuse retrogradely into presynaptic terminals, contributing to the induction of hippocampal long-term potentiation (LTP). Here, we present novel evidence that NO is required for kainate receptor (KAR)-dependent presynaptic form of LTP (pre-LTP) in the adult insular cortex (IC). In the IC, we found that inhibition of NO synthase erased the maintenance of pre-LTP, while the induction of pre-LTP required the activation of KAR. Furthermore, NO is essential for pre-LTP induced between two pyramidal cells in the IC using the double patch-clamp recording. These results suggest that NO is required for homosynaptic pre-LTP in the IC. Our results present strong evidence for the critical roles of NO in pre-LTP in the IC. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Ready-to-Record Cells for Kinetic Screening of VGICs.

Voltage-gated ion channels (VGICs) are integral membrane proteins crucial for transmitting electrical signals in excitable cells. Understanding the kinetics of these ion channels requires conducting patch-clamp experiments using genetically modified cell lines that express a single type of ion channel gene. However, this process relies on the continuous maintenance of cell lines to ensure an adequate supply of sample cells for patch-clamp experiments. Advancements in automated patch-clamp methods have enabled researchers to significantly increase the number of patch-clamped cells per experiment, from just a few cells to as many as 384 cells. Despite this progress, the manual task of preparing the cell samples remains a significant bottleneck in the kinetic screening of VGICs. Here we describe a method to address this challenge by generating ready-to-record (RTR) VGIC-expressing cells that can be frozen and stored separately from patch-clamp experiments. This decoupling of the cell sample preparation process from the patch-clamp experiments offers a streamlined approach to studying VGICs on manual or an automated patch-clamp system.

Voltage Clamp Fluorometry: Illuminating the Dynamics of Ion Channels.

Ion channels comprise one of the largest targets for drug development and treatment and have been a subject of enduring fascination since first discovered in the 1950s. Over the past decades, thousands of publications have explored the cellular biology and molecular physiology of these proteins, and many channel structures have been determined since the late 1990s. Trying to connect the dots between ion channel function and structure, voltage clamp fluorometry (VCF) emerges as a powerful tool because it allows monitoring of the conformational rearrangements underlying the different functional states of the channel. This technique represents an elegant harmonization of molecular biology, electrophysiology, and fluorescence. In the following chapter, we will provide a concise guide to performing VCF on Xenopus laevis oocytes using the two-electrode voltage clamp (TEVC) modality. This is the most widely used configuration on Xenopus oocytes for its relative simplicity and demonstrated success in a number of different ion channels utilizing a variety of attached labels.

The Dynamic Clamp Technique: A Robust Toolkit for Investigating Potassium Channel Function.

The dynamic clamp technique has emerged as a powerful tool in the field of cardiac electrophysiology, enabling researchers to investigate the intricate dynamics of ion currents in cardiac cells. Potassium channels play a critical role in the functioning of cardiac cells and the overall electrical stability of the heart. This chapter provides a comprehensive overview of the methods and applications of dynamic clamp in the study of key potassium currents in cardiac cells. A step-by-step guide is presented, detailing the experimental setup and protocols required for implementing the dynamic clamp technique in cardiac cell studies. Special attention is given to the design and construction of a dynamic clamp setup with Real Time eXperimental Interface, configurations, and the incorporation of mathematical models to mimic ion channel behavior. The chapter's core focuses on applying dynamic clamp to elucidate the properties of various potassium channels in cardiac cells. It discusses how dynamic clamp can be used to investigate channel kinetics, voltage-dependent properties, and the impact of different potassium channel subtypes on cardiac electrophysiology. The chapter will also include examples of specific dynamic clamp experiments that studied potassium currents or their applications in cardiac cells.

Machine Learning Methods for the Analysis of the Patch-Clamp Signals.

Patch-clamp technique provides a unique possibility to record the ion channels' activity. This method enables tracking the changes in their functional states at controlled conditions on a real-time scale. Kinetic parameters evaluated for the patch-clamp signals form the fundamentals of electrophysiological characteristics of the channel functioning. Nevertheless, the noisy series of ionic currents flowing through the channel protein(s) seem to be bountiful of information, and the standard data processing techniques likely unravel only its part. Rapid development of artificial intelligence (AI) techniques, especially machine learning (ML), gives new prospects for whole channelology. Here we consider the question of the AI applications in the patch-clamp signal analysis. It turns out that the AI methods may not only enable for automatizing of signal analysis, but also they can be used in finding inherent patterns of channel gating and allow the researchers to uncover the details of gating machinery, which had been never considered before. In this work, we outline the currently known AI methods that turned out to be utilizable and useful in the analysis of patch-clamp signals. This chapter can be considered an introductory guide to the application of AI methods in the analysis of the time series of channel currents (together with its advantages, disadvantages, and limitations), but we also propose new possible directions in this field.

Structure-Activity Relationship Studies in a Series of Xanthine Inhibitors of SLACK Potassium Channels.

Gain-of-function mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel known as SLACK, are associated with the rare but devastating developmental and epileptic encephalopathy known as epilepsy of infancy with migrating focal seizures (EIMFS). The design of small molecule inhibitors of SLACK channels represents a potential therapeutic approach to the treatment of EIMFS, other childhood epilepsies, and developmental disorders. Herein, we describe a hit optimization effort centered on a xanthine SLACK inhibitor (8) discovered via a high-throughput screen. Across three distinct regions of the chemotype, we synthesized 58 new analogs and tested each one in a whole-cell automated patch-clamp assay to develop structure-activity relationships for inhibition of SLACK channels. We further evaluated selected analogs for their selectivity versus a variety of other ion channels and for their activity versus clinically relevant SLACK mutants. Selectivity within the series was quite good, including versus hERG. Analog 80 (VU0948578) was a potent inhibitor of WT, A934T, and G288S SLACK, with IC50 values between 0.59 and 0.71 µM across these variants. VU0948578 represents a useful in vitro tool compound from a chemotype that is distinct from previously reported small molecule inhibitors of SLACK channels.

The metamorphosis of amphibian myocardium: moving to the heart of the matter.

Amphibians are a classical object for physiological studies, and they are of great value for developmental studies owing to their transition from an aquatic larval form to an adult form with a terrestrial lifestyle. Axolotls (Ambystoma mexicanum) are of special interest for such studies because of their neoteny and facultative pedomorphosis, as in these animals, metamorphosis can be induced and fully controlled in laboratory conditions. It has been suggested that their metamorphosis, associated with gross anatomical changes in the heart, also involves physiological and electrical remodeling of the myocardium. We used whole-cell patch clamp to investigate possible changes caused by metamorphosis in electrical activity and major ionic currents in cardiomyocytes isolated from paedomorphic and metamorphic axolotls. T4-induced metamorphosis caused shortening of atrial and ventricular action potentials (APs), with no changes in resting membrane potential or maximum velocity of AP upstroke, favoring higher heart rate possible in metamorphic animals. Potential-dependent potassium currents in axolotl myocardium were represented by delayed rectifier currents IKr and IKs, and upregulation of IKs caused by metamorphosis probably underlies AP shortening. Metamorphosis was associated with downregulation of inward rectifier current IK1, probably serving to increase the excitability of myocardium in metamorphic animals. Metamorphosis also led to a slight increase in fast sodium current INa with no changes in its steady-state kinetics and to a significant upregulation of ICa in both atrial and ventricular cells, indicating stronger Ca2+ influx for higher cardiac contractility in metamorphic salamanders. Taken together, these changes serve to increase cardiac reserve in metamorphic animals.

Scanless two-photon voltage imaging.

Two-photon voltage imaging has long been heralded as a transformative approach capable of answering many long-standing questions in modern neuroscience. However, exploiting its full potential requires the development of novel imaging approaches well suited to the photophysical properties of genetically encoded voltage indicators. We demonstrate that parallel excitation approaches developed for scanless two-photon photostimulation enable high-SNR two-photon voltage imaging. We use whole-cell patch-clamp electrophysiology to perform a thorough characterization of scanless two-photon voltage imaging using three parallel illumination approaches and lasers with different repetition rates and wavelengths. We demonstrate voltage recordings of high-frequency spike trains and sub-threshold depolarizations from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-Kv. Using a low repetition-rate laser, we perform multi-cell recordings from up to fifteen targets simultaneously. We co-express JEDI-2P-Kv and the channelrhodopsin ChroME-ST and capitalize on their overlapping two-photon absorption spectra to simultaneously evoke and image action potentials using a single laser source. We also demonstrate in vivo scanless two-photon imaging of multiple cells simultaneously up to 250 µm deep in the barrel cortex of head-fixed, anaesthetised mice.

Hippocampal-dependent navigation in head-fixed mice using a floating real-world environment.

Head-fixation of mice enables high-resolution monitoring of neuronal activity coupled with precise control of environmental stimuli. Virtual reality can be used to emulate the visual experience of movement during head fixation, but a low inertia floating real-world environment (mobile homecage, MHC) has the potential to engage more sensory modalities and provide a richer experimental environment for complex behavioral tasks. However, it is not known whether mice react to this adapted environment in a similar manner to real environments, or whether the MHC can be used to implement validated, maze-based behavioral tasks. Here, we show that hippocampal place cell representations are intact in the MHC and that the system allows relatively long (20 min) whole-cell patch clamp recordings from dorsal CA1 pyramidal neurons, revealing sub-threshold membrane potential dynamics. Furthermore, mice learn the location of a liquid reward within an adapted T-maze guided by 2-dimensional spatial navigation cues and relearn the location when spatial contingencies are reversed. Bilateral infusions of scopolamine show that this learning is hippocampus-dependent and requires intact cholinergic signalling. Therefore, we characterize the MHC system as an experimental tool to study sub-threshold membrane potential dynamics that underpin complex navigation behaviors.

Cell size induced bias of current density in hypertrophic cardiomyocytes.

Alterations in ion channel expression and function known as "electrical remodeling" contribute to the development of hypertrophy and to the emergence of arrhythmias and sudden cardiac death. However, comparing current density values - an electrophysiological parameter commonly utilized to assess ion channel function - between normal and hypertrophied cells may be flawed when current amplitude does not scale with cell size. Even more, common routines to study equally sized cells or to discard measurements when large currents do not allow proper voltage-clamp control may introduce a selection bias and thereby confound direct comparison. To test a possible dependence of current density on cell size and shape, we employed whole-cell patch-clamp recording of voltage-gated sodium and calcium currents in Langendorff-isolated ventricular cardiomyocytes and Purkinje myocytes, as well as in cardiomyocytes derived from trans-aortic constriction operated mice. Here, we describe a distinct inverse relationship between voltage-gated sodium and calcium current densities and cell capacitance both in normal and hypertrophied cells. This inverse relationship was well fit by an exponential function and may be due to physiological adaptations that do not scale proportionally with cell size or may be explained by a selection bias. Our study emphasizes the need to consider cell size bias when comparing current densities in cardiomyocytes of different sizes, particularly in hypertrophic cells. Conventional comparisons based solely on mean current density may be inadequate for groups with unequal cell size or non-proportional current amplitude and cell size scaling.