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A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K + (K ATP ) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the K ATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys 166 prevented these effects. Clinical variants in KCNJ11 that affect Cys 166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP 2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys 166 enhanced the PIP 2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of K ATP channels by α1-adrenoreceptor stimulation. The Cys 166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels., Competing Interests: The authors declare no competing interest.

The norepinephrine-releasing neurons in the locus coeruleus (LC) are well known to regulate wakefulness/arousal. They display active firing during wakefulness and a decreased discharge rate during sleep. We have previously reported that LC neurons express large numbers of GABA B receptors (GABA B Rs) located at peri-/extrasynaptic sites and are subject to tonic inhibition due to the continuous activation of GABA B Rs by ambient GABA, which is significantly higher during sleep than during wakefulness. In this study, we further showed using western blot analysis that the activation of GABA B Rs with baclofen could increase the level of phosphorylated extracellular signal-regulated kinase 1 (ERK 1 ) in LC tissue. Recordings from LC neurons in brain slices showed that the inhibition of ERK 1/2 with U0126 and FR180204 accelerated the decay of whole-cell membrane current induced by prolonged baclofen application. In addition, the inhibition of ERK 1/2 also increased spontaneous firing and reduced tonic inhibition of LC neurons after prolonged exposure to baclofen. These results suggest a new role of GABA B Rs in mediating ERK 1 -dependent autoregulation of the stability of GABA B R-activated whole-cell current, in addition to its well-known effect on gated potassium channels, to cause a tonic current in LC neurons.

Mechanosensitive (MS) channels have an intimate relationship with membrane lipids that underlie their mechanosensitivity. Membrane lipids may influence channel activity by directly interacting with MS channels or by influencing the global properties of the membrane such as elastic area expansion modulus or bending rigidity. Previous work has implicated membrane stiffness as a potential determinant of the mechanosensitivity of E. coli (Ec)MscS. Here we systematically tested this hypothesis using patch fluorometry of azolectin liposomes doped with lipids of increasing elastic area expansion modulus. Increasing dioleoylphosphatidylethanolamine (DOPE) content of azolectin liposomes made it more difficult to activate EcMscS by membrane tension (i.e. increased gating threshold). This effect was exacerbated by stiffer forms of phosphatidylethanolamine such as the branched chain lipid diphytanoylphosphoethanolamine (DPhPE) or the fully saturated lipid distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). Furthermore, a comparison of the branched chain lipid diphytanoylphosphocholine (DPhPC) to the stiffer DPhPE indicated again that it was harder to activate EcMscS in the presence of the stiffer DPhPE. We show that these effects are not due to changes in membrane bending rigidity as the membrane tension threshold of EcMscS in membranes doped with PC18:1 and PC18:3 remained the same, despite a two-fold difference in their bending rigidity. We also show that after prolonged pressure application sudden removal of force in softer membranes caused a rebound reactivation of EcMscS and we discuss the relevance of this phenomenon to bacterial osmoregulation. Collectively, our data suggests that membrane stiffness (elastic area expansion modulus) is one of the key determinants of the mechanosensitivity of EcMscS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The electrophysiological properties of pulmonary vein (PV)-cardiomyocytes, and their responses to the sympathetic neurotransmitter, noradrenaline (NA), are thought to differ from those of the left atrium (LA) and contribute to atrial ectopy. The aim of this study was to examine rat PV cardiomyocyte electrophysiology and responses to NA in comparison with LA cells. LA and PV cardiomyocytes were isolated from adult male Wistar rat hearts, and membrane potentials and ion currents recorded at 36°C using whole-cell patch-clamp techniques. PV and LA cardiomyocytes did not differ in size. In control, there were no differences between the two cell-types in zero-current potential or action potential duration (APD) at 1 Hz, although the incidence of early afterdepolarizations (EADs) was greater in PV than LA cardiomyocytes. The L-type Ca 2+ current (I CaL ) was ~×1.5 smaller (p = .0029, Student's t test) and the steady-state K + current (I Kss ) was ~×1.4 larger (p = .0028, Student's t test) in PV than in LA cardiomyocytes. PV cardiomyocyte inward-rectifier current (I K1 ) was slightly smaller than LA cardiomyocyte I K1 . In LA cardiomyocytes, NA significantly prolonged APD 30 . In PV cells, APD 30 responses to 1 μM NA were heterogeneous: while the mean percentage change in APD 30 was not different from 0 (16.5 ± 9.7%, n cells/N animals = 12/10, p = .1177, one-sample t test), three cells showed shortening (-18.8 ± 6.0%) whereas nine showed prolongation (28.3 ± 10.1%, p = .008, Student's t test). NA had no effect on I K1 in either cell-type but inhibited PV I Kss by 41.9 ± 4.1% (n/N = 23/11 p < .0001), similar to LA cells. NA increased I CaL in most PV cardiomyocytes (median × 2.2-increase, p < .0001, n/N = 32/14, Wilcoxon-signed-rank test), although in 7/32 PV cells I CaL was decreased following NA. PV cardiomyocytes differ from LA cells and respond heterogeneously to NA., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Introduction: During mammalian brain development, neural activity leads to maturation of glutamatergic innervations to locus coeruleus. In this study, fast excitatory postsynaptic currents mediated by N-methyl-d-aspartate (NMDA) receptors were evaluated to investigate the maturation of excitatory postsynaptic currents in locus coeruleus (LC) neurons., Methods: NMDA receptor-mediated synaptic currents in LC neurons were evaluated using whole-cell voltage-clamp recording during the primary postnatal weeks. This technique was used to calculate the optimum holding potential for NMDA receptor-mediated currents and the best frequency for detecting spontaneous excitatory postsynaptic currents (sEPSC)., Results: The optimum holding potential for detecting NMDA receptor-mediated currents was + 40 to + 50 mV in LC neurons. The frequency, amplitude, rise time, and decay time constant of synaptic responses depended on the age of the animal and increased during postnatal maturation., Conclusion: These findings suggest that most nascent glutamatergic synapses express functional NMDA receptors in the postnatal coerulear neurons, and that the activities of the neurons in this region demonstrate an age-dependent variation.

Despite the wide application of carvacrol (CAR) in medicines, dietary supplements, and foods, there is still insufficient electrophysiological data on the mechanisms of action of CAR, particularly with regard to heart function. Therefore, in this study, we attempted to elucidate whether CAR, whose inhibitory effect on both cardiac and vascular TRPM7 and L-type Ca 2+ currents has been demonstrated previously, could modify cardiac electrical activity. We used a combination of optical mapping and microelectrode techniques to track the action potentials (APs) and the spread of electrical activity in a Langendorff-perfused rabbit heart model during atrial/endo/epicardial pacing. Simultaneously, ECG recordings were acquired. Because human trials on CAR are still lacking, we tested the action of CAR on human ventricular preparations obtained from explanted hearts. Activation time (AT), AP duration (APD), and conduction velocity maps were constructed. We demonstrated that at a low concentration (10  μ M) of CAR, only marginal changes in the AP parameters were observed. At higher concentrations (≥100  μ M), a decrease in AP upstroke velocity ( dV / dt max ), suggesting inhibition of Na + current, and APD (at 50 and 90% repolarization) was detected; also slowing in the spread of electrical signals via the atrioventricular node was observed, suggesting impaired functioning of Ca 2+ channels. In addition, a decrease in the T-wave amplitude was seen on the ECG, suggesting an impaired repolarization process. Nevertheless, those changes occurred without a significant impact on the resting membrane potential and were reversible. We suggest that CAR might play a role in modulating cardiac electrical activity at high concentrations., Competing Interests: The authors declare that they have no financial interests or potential conflicts of interest., (Copyright © 2020 Mantė Almanaitytė et al.)

Aim: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca 2+ entry through voltage-dependent Ca 2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca 2+ current and intracellular Ca 2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells., Methods: Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca 2+ was estimated using epifluorescence microscopy and fluo-8 (salt form)., Results: Cells stimulated by brief depolatizations in absence of extracellular Ca +2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca +2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca 2+ channel gating), (ii) in cells from knock-out P/Q-type Ca 2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association)., Conclusion: We demonstrated that Ca 2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca 2+ channels can operate as voltage sensors of this process., (© 2019 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of caffeic acid phenethyl ester (CAPE) on the pacemaker potentials of ICCs from the mouse small or large intestine. Using the whole-cell patch-clamp configuration, we found that CAPE depolarized the pacemaker potentials of cultured ICCs from the murine small intestine in a dose-dependent manner. The estrogen receptor (ER) β antagonist PHTPP completely inhibited CAPE-induced depolarization, but the ERα antagonist BHPI did not. Intracellular GDP-β-S and pretreatment with Ca 2+ -free solution or thapsigargin also blocked CAPE-induced depolarization. To investigate the mechanisms of CAPE-mediated depolarization of ICCs, we used the nonselective cation channel (NSCC) inhibitor flufenamic acid, the Cl - channel blocker, mitogen-activated protein kinase (MAPK) inhibitors PD98059, SB203580, or SP600125, and PI3 kinase inhibitor LY294002. All inhibitors blocked the CAPE-induced pacemaker potential depolarization of ICCs. These results suggest that CAPE induces pacemaker potential depolarization through ERβ in a G protein, NSCC, Cl - channel, MAPK- and PI3 kinase dependent manner via intracellular and extracellular Ca 2+ regulation in the murine small intestine. CAPE may therefore modulate GI motility by acting on ICCs in the murine small intestine.

During the critical period, neuronal connections are shaped by sensory experience. While the basis for this temporarily heightened plasticity remains unclear, shared connections introducing activity correlations likely play a key role. Thus, we investigated the changing intracortical connectivity in primary auditory cortex (A1) over development. In adult, layer 2/3 (L2/3) neurons receive ascending inputs from layer 4 (L4) and also receive few inputs from subgranular layer 5/6 (L5/6). We measured the spatial pattern of intracortical excitatory and inhibitory connections to L2/3 neurons in slices of mouse A1 across development using laser-scanning photostimulation. Before P11, L2/3 cells receive most excitatory input from within L2/3. Excitatory inputs from L2/3 and L4 increase after P5 and peak during P9-16. L5/6 inputs increase after P5 and provide most input during P12-16, the peak of the critical period. Inhibitory inputs followed a similar pattern. Functional circuit diversity in L2/3 emerges after P16. In vivo two-photon imaging shows low pairwise signal correlations in neighboring neurons before P11, which peak at P15-16 and decline after. Our results suggest that the critical period is characterized by high pairwise activity correlations and that transient hyperconnectivity of specific circuits, in particular those originating in L5/6, might play a key role., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Purpose: In the mammalian retina, cannabinoid type 1 receptors (CB1Rs) are well-positioned to alter inhibitory synaptic function from amacrine cells and, thus, might influence visual signal processing in the inner retina. However, it is not known if CB1R modulates amacrine cells feedback inhibition at retinal bipolar cell (BC) terminals., Methods: Using whole-cell voltage-clamp recordings, we examined the pharmacological effect of CB1R activation and inhibition on spontaneous inhibitory postsynaptic currents (sIPSCs) and glutamate-evoked IPSCs (gIPSCs) from identified OFF BCs in light-adapted rat retinal slices., Results: Activation of CB1R with WIN55212-2 selectively increased the frequency of GABAergic, but not glycinergic sIPSC in types 2, 3a, and 3b OFF BCs, and had no effect on inhibitory activity in type 4 OFF BCs. The increase in GABAergic activity was eliminated in axotomized BCs and can be suppressed by blocking CB1R with AM251 or GABAA and GABAρ receptors with SR-95531 and TPMPA, respectively. In all OFF BC types tested, a brief application of glutamate to the outer plexiform layer elicited gIPSCs comprising GABAergic and glycinergic components that were unaffected by CB1R activation. However, blocking CB1R selectively increased GABAergic gIPSCs, supporting a role for endocannabinoid signaling in the regulation of glutamate-evoked GABAergic inhibitory feedback to OFF BCs., Conclusions: CB1R activation shape types 2, 3a, and 3b OFF BC responses by selectively regulate GABAergic feedback inhibition at their axon terminals, thus cannabinoid signaling might play an important role in the fine-tuning of visual signal processing in the mammalian inner retina.

Current in vitro assays typically poorly predict cardiac liability as they focus on single ion channels overexpressed in cell lines. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), on the other hand, provide a unique opportunity for drug testing on human cardiomyocytes using high-throughput systems. However, these cells can differ from adult cardiomyocytes in their ion channel expression and, therefore, electrophysiologic properties. One of the main challenges of hiPSC-CMs is the physiologic expression of ion channels such as the inward rectifiers (e.g., Kir2.1-2.3), which conduct the cardiac inward rectifier potassium current (I K1 ). I K1 is one of the primary contributors in maintaining a stable resting membrane potential in cardiac cells, which is essential for excitability. This is only expressed in low levels, or sometimes not at all, in hiPSC-CMs as shown by patch clamp studies. Dynamic clamp is a method of electronically introducing ion currents (e.g., I K1 ) into cells to compensate for the lack of endogenous expression, thus offering the potential to record more stable action potentials in hiPSC-CMs. In this article, we describe the method of using hiPSC-CMs on an automated patch clamp device (Patchliner) coupled with the automated dynamic clamp add-on (Dynamite 8 ). We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell-derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated I K1 and compensate seal resistance Support Protocol 1: Cardiomyocyte plating and culture Support Protocol 2: Cell harvesting and dissociation Alternate Protocol: Recording action potential pharmacology at physiologic temperatures., (© 2019 John Wiley & Sons, Inc.)

Voltage-gated calcium-channels (VGCCs) are heteromers consisting of several subunits. Mutations in the genes coding for VGCC subunits have been reported to be associated with autism spectrum disorder (ASD). In a previous study, we identified electrophysiologically relevant missense mutations of Ca V β 2 subunits of VGCCs. From this, we derived the hypothesis that several Ca V β 2 -mutations associated with ASD show common features sensitizing LTCCs and/or enhancing currents. Using a Ca V β 2d backbone, we performed extensive whole-cell and single-channel patch-clamp analyses of Ba 2+ currents carried by Ca v 1.2 pore subunits co-transfected with the previously described Ca V β 2 mutations (G167S, S197F) as well as a recently identified point mutation (V2D). Furthermore, the interaction of the mutated Ca V β 2d subunits with the RGK protein Gem was analyzed by co-immunoprecipitation assays and electrophysiological studies. Patch-clamp analyses revealed that all mutations increase Ba 2+ currents, e.g. by decreasing inactivation or increasing fraction of active sweeps. All Ca V β 2 mutations interact with Gem, but differ in the extent and characteristics of modulation by this RGK protein (e.g. decrease of fraction of active sweeps: Ca V β 2d&#95;G167S > Ca V β 2d&#95;V2D > Ca V β 2d&#95;S197F ). In conclusion, patch-clamp recordings of ASD-associated Ca V β 2d mutations revealed differential modulation of Ba 2+ currents carried by Ca V 1.2 suggesting kind of an "electrophysiological fingerprint" each. The increase in current finally observed with all Ca V β 2d mutations analyzed might contribute to the complex pathophysiology of ASD and by this indicate a possible underlying molecular mechanism., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Automated patch-clamp (APC) systems have become indispensable tools of drug-discovery programs by allowing high-throughput electrophysiology-based screening of ion channel compounds. The recent development and introduction of microfluidics-based APC systems have made it possible to study the interactions of ligand-gated ion channels with pharmacological reagents, such as agonists, antagonists, or positive allosteric modulators (PAMs), with reliable pharmacological results comparable to those of the gold-standard manual patch-clamp technique while maintaining high-throughput capacity. Many ligand-gated ion channels exhibit rapid desensitization upon repetitive introduction of ligands; this loss of channel activity in the absence of pharmacological interaction poses a challenge for developing accurate, precise, and robust assays with high success rate, low run-down, and reliable pharmacological results. Here we present procedures to study nicotinic acetylcholine receptors (nAChRs) with the IonFlux™, an automated patch-clamp system with continuous flow and precise fluidic exchange; these procedures can also be generalized to the study of other ligand-gated ion channels. We present protocols to study agonist, antagonist, and PAM activities on nAChRs, particularly the rapidly desensitizing nAChR α7 receptors. The data demonstrate that the IonFlux™ system is a fast, robust, and reliable platform for the study of nAChRs and other ligand-gated ion channels, generating data that closely mimic those from manual patch-clamp conditions. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring agonist concentration-dependent response Basic Protocol 2: Measuring antagonist concentration-dependent response Basic Protocol 3: Measuring positive allosteric modulator (PAM) concentration-dependent response Support Protocol 1: Basic IonFlux system operation Support Protocol 2: Plate care and filling Support Protocol 3: Plate preparation for water rinsing Support Protocol 4: Water rinsing of plates Support Protocol 5: Plate priming Support Protocol 6: General assay Support Protocol 7: Editing the compound addition sequence (compound list) Support Protocol 8: Creating compound list for agonist concentration-dependent response Support Protocol 9: Creating compound list for antagonist or PAM concentration-dependent response Support Protocol 10: Defining the different compounds used or compound list Support Protocol 11: Maintenance Support Protocol 12: Data analysis Support Protocol 13: Cell culture., (© 2020 John Wiley & Sons, Inc.)

Aims: Detect the antiarrhythmic effect of crotonoside (Cro)., Main Methods: We used whole-cell patch-clamp techniques to detect the effects of Cro on action potentials (APs) and transmembrane ion currents in isolated rabbit left ventricular myocytes. We also verified the effect of Cro on ventricular arrhythmias caused by aconitine in vivo., Key Findings: Cro reduced the maximum depolarization velocity (V max ) of APs and shortened the action potential duration (APD) in a concentration-dependent manner, but it had no significant effect on the resting membrane potential (RMP) or action potential amplitude (APA). It also inhibited the peak sodium current (I Na ) and L-type calcium current (I CaL ) in a concentration-dependent manner with half-maximal inhibitory concentrations (IC 50 ) of 192 μmol/L and 159 μmol/L, respectively. However, Cro had no significant effects on the inward rectifier potassium current (I K1 ) or rapidly activating delayed rectifier potassium current (I Kr ). Sea anemone toxin II (ATX II) increased the late sodium current (I NaL ), but Cro abolished this effect. Moreover, Cro significantly abolished ATX II-induced early afterdepolarizations (EADs) and high extracellular Ca 2+ concentration (3.6 mmol/L)-induced delayed afterdepolarizations (DADs). We also verified that Cro effectively delayed the onset time and reduced the incidence of ventricular arrhythmias caused by aconitine in vivo., Significance: These results revealed that Cro effectively inhibits I Na , I NaL , and I CaL in ventricular myocytes. Cro has antiarrhythmic potential and thus deserves further study., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Most single cell RNA sequencing protocols start with single cells dispersed from intact tissue. High-throughput processing of the separated cells is enabled using microfluidics platforms. However, dissociation of tissue results in loss of information about cell location and morphology and potentially alters the transcriptome. An alternative approach for collecting RNA from single cells is to re-purpose the electrophysiological technique of patch clamp recording. A hollow patch pipette is attached to individual cells, enabling the recording of electrical activity, after which the cytoplasm may be extracted for single cell RNA-Seq ("Patch-Seq"). Since the tissue is not disaggregated, the location of cells is readily determined, and the morphology of the cells is maintained, making possible the correlation of single cell transcriptomes with cell location, morphology and electrophysiology. Recent Patch-Seq studies utilizes PCR amplification to increase amount of nucleic acid material to the level required for current sequencing technologies. PCR is prone to create biased libraries - especially with the extremely high degrees of exponential amplification required for single cell amounts of RNA. We compared a PCR-based approach with linear amplifications and demonstrate that aRNA amplification (in vitro transcription, IVT) is more sensitive and robust for single cell RNA collected by a patch clamp pipette.

Cortical layer 1 (L1) interneurons have been proposed as a hub for attentional modulation of underlying cortex, but the transformations that this circuit implements are not known. We combined genetically targeted voltage imaging with optogenetic activation and silencing to study the mechanisms underlying sensory processing in mouse barrel cortex L1. Whisker stimuli evoked precisely timed single spikes in L1 interneurons, followed by strong lateral inhibition. A mild aversive stimulus activated cholinergic inputs and evoked a bimodal distribution of spiking responses in L1. A simple conductance-based model that only contained lateral inhibition within L1 recapitulated the sensory responses and the winner-takes-all cholinergic responses, and the model correctly predicted that the network would function as a spatial and temporal high-pass filter for excitatory inputs. Our results demonstrate that all-optical electrophysiology can reveal basic principles of neural circuit function in vivo and suggest an intuitive picture for how L1 transforms sensory and modulatory inputs. VIDEO ABSTRACT., Competing Interests: Declaration of Interests A.E.C. is a founder of Q-State Biosciences and is on the scientific advisory board. A.E.C., L.Z.F., and S.K. have filed a patent on the imaging system., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Structure-activity relationship studies of a reported menthol-based transient receptor potential cation channel subfamily M member 8 channel (TRPM8) antagonist, guided by computational simulations and structure-based design, uncovers a novel series of TRPM8 antagonists with >10-fold selectivity versus related TRP subtypes. Spiro[4.5]decan-8-yl analogue 14 inhibits icilin-evoked Ca 2+ entry in HEK-293 cells stably expressing human TRPM8 (hTRPM8) with an IC 50 of 2.4 ± 1.0 nM, while in whole-cell patch-clamp recordings this analogue inhibits menthol-evoked currents with a hTRPM8 IC 50 of 64 ± 2 nM. Molecular dynamics (MD) simulations of compound 14 in our homology model of hTRPM8 suggest that this antagonist forms extensive hydrophobic contacts within the orthosteric site. In the wet dog shakes (WDS) assay, compound 14 dose-dependently blocks icilin-triggered shaking behaviors in mice. Upon local administration, compound 14 dose dependently inhibits cold allodynia evoked by the chemotherapy oxaliplatin in a murine model of peripheral neuropathy at microgram doses. Our findings suggest that 14 and other biphenyl amide analogues within our series can find utility as potent antagonist chemical probes derived from (-)-menthol as well as small molecule therapeutic scaffolds for chemotherapy-induced peripheral neuropathy (CIPN) and other sensory neuropathies.

Trimeric acid-sensing ion channels (ASICs) contribute to neuronal signaling by converting extracellular acidification into excitatory sodium currents. Previous work with homomeric ASIC1a implicates conserved leucine (L7') and consecutive glycine-alanine-serine (GAS belt) residues near the middle, and conserved negatively charged (E18') residues at the bottom of the pore in ion permeation and/or selectivity. However, a conserved mechanism of ion selectivity throughout the ASIC family has not been established. We therefore explored the molecular determinants of ion selectivity in heteromeric ASIC1a/ASIC2a and homomeric ASIC2a channels using site-directed mutagenesis, electrophysiology, and molecular dynamics free energy simulations. Similar to ASIC1a, E18' residues create an energetic preference for sodium ions at the lower end of the pore in ASIC2a-containing channels. However, and in contrast to ASIC1a homomers, ion permeation through ASIC2a-containing channels is not determined by L7' side chains in the upper part of the channel. This may be, in part, due to ASIC2a-specific negatively charged residues (E59 and E62) that lower the energy of ions in the upper pore, thus making the GAS belt more important for selectivity. This is confirmed by experiments showing that the L7'A mutation has no effect in ASIC2a, in contrast to ASIC1a, where it eliminated selectivity. ASIC2a triple mutants eliminating both L7' and upper charges did not lead to large changes in selectivity, suggesting a different role for L7' in ASIC2a compared with ASIC1a channels. In contrast, we observed measurable changes in ion selectivity in ASIC2a-containing channels with GAS belt mutations. Our results suggest that ion conduction and selectivity in the upper part of the ASIC pore may differ between subtypes, whereas the essential role of E18' in ion selectivity is conserved. Furthermore, we demonstrate that heteromeric channels containing mutations in only one of two ASIC subtypes provide a means of functionally testing mutations that render homomeric channels nonfunctional., (© 2020 Lynagh et al.)

Voltage gated sodium channel (VGSC) activation drives the action potential upstroke in cardiac myocytes, skeletal muscles, and neurons. After opening, VGSCs rapidly enter a non-conducting, inactivated state. Impaired inactivation causes persistent inward current and underlies cardiac arrhythmias. VGSC auxiliary proteins calmodulin (CaM) and fibroblast growth factor homologous factors (FHFs) bind to the channel's C-terminal domain (CTD) and limit pathogenic persistent currents. The structural details and mechanisms mediating these effects are not clear. Building on recently published cryo-EM structures, we show that CaM and FHF limit persistent currents in the cardiac NaV1.5 VGSC by stabilizing an interaction between the channel's CTD and III-IV linker region. Perturbation of this intramolecular interaction increases persistent current and shifts the voltage dependence of steady-state inactivation. Interestingly, the NaV1.5 residues involved in the interaction are sites mutated in the arrhythmogenic long QT3 syndrome (LQT3). Along with electrophysiological investigations of this interaction, we present structural models that suggest how CaM and FHF stabilize the interaction and thereby limit the persistent current. The critical residues at the interaction site are conserved among VGSC isoforms, and subtle substitutions provide an explanation for differences in inactivation among the isoforms., (© 2019 Gade et al.)

Diminazene aceturate (DIZE) is an anti-protozoan compound that has been previously reported to increase the activity of the angiotensin-converting enzyme 2 (ACE2) and thus increase Angiotensin-(1-7) production, leading to cardioprotection against post-myocardial infarction dysfunction and structural remodelling. Moreover, DIZE is able to ameliorate morpho-functional changes after myocardial infarction by enhancing ACE2 activity, thus increasing Angiotensin-(1-7) production (a benefic peptide of the renin-angiotensin system). However, despite the improvement in cardiac function/structure, little is known about DIZE effects on arrhythmia suppression, contraction/excitable aspects of the heart and importantly its mechanisms of action. Thus, our aim was to test the acute effect of DIZE cardioprotection at the specific level of potential antiarrhythmic effects and modulation in excitation-contraction coupling. For this, we performed in vitro and in vivo techniques for arrhythmia induction followed by an acute administration of DIZE. For the first time, we described that DIZE can reduce arrhythmias which is explained by modulation of cardiomyocyte contraction and excitability. Such effects were independent of Mas receptor and nitric oxide release. Development of a new DIZE-based approach to ameliorate myocardial contractile and electrophysiological dysfunction requires further investigation; however, DIZE may provide the basis for a future beneficial therapy to post-myocardial infarction patients., (© 2019 John Wiley & Sons Australia, Ltd.)

Nonselective cation channels, consistent with transient receptor potential melastatin-4 (TRPM4), regulate detrusor smooth muscle (DSM) function. TRPM4 channels can exist as homomers or assemble with sulfonylurea receptors (SURs) as complexes. We evaluated contributions of TRPM4/SUR-TRPM4 channels to DSM excitability and contractility by examining the effects of TRPM4/SUR-TRPM4 channel modulators 9-phenanthrol, glibenclamide, and diazoxide on freshly-isolated guinea pig DSM cells (amphotericin-B perforated patch-clamp electrophysiology) and mucosa-free DSM strips (isometric tension recordings). In DSM cells, complete removal of extracellular Na + decreased voltage-step-induced cation (non-K + selective) currents. At high positive membrane potentials, 9-phenanthrol at 100 μM attenuated voltage step-induced currents more effectively than at 30 μM, revealing concentration-dependent, voltage-sensitive inhibition. In comparison to 9-phenanthrol, glibenclamide (100 μM) displayed lower inhibition of cation currents. In the presence of glibenclamide (100 μM), 9-phenanthrol (100 μM) further decreased the currents. The SUR-TRPM4 complex activator diazoxide (100-300 μM) weakly inhibited the currents. 9-Phenanthrol, but not glibenclamide or diazoxide, increased cell capacitance (a cell surface area indicator). In contractility studies, glibenclamide displayed lower potencies than 9-phenanthrol attenuating spontaneous and 20 mM KCl-induced DSM phasic contractions. While both compounds showed similar maximum inhibitions on DSM spontaneous phasic contractions, glibenclamide was generally less efficacious on 20 mM KCl-induced phasic contractions. In summary, the observed differential effects of 9-phenanthrol and glibenclamide on DSM excitability and contractility support unique mechanisms for the two compounds. The data suggest that SUR-TRPM4 complexes do not contribute to DSM function. This study advances our understanding of pharmacological effects of glibenclamide and 9-phenanthrol on DSM cell cation currents.

Aims: The mode of action by which doxapram acts as a respiratory stimulant in humans is controversial. Studies in rodent models, have shown that doxapram is a more potent and selective inhibitor of TASK-1 and TASK-1/TASK-3 heterodimer channels, than TASK-3. Here we investigate the direct effect of doxapram and chirally separated, individual positive and negative enantiomers of the compound, on both human and mouse, homodimeric and heterodimeric variants of TASK-1 and TASK-3., Methods: Whole-cell patch clamp electrophysiology on tsA201 cells was used to assess the potency of doxapram on cloned human or mouse TASK-1, TASK-3 and TASK-2 channels. Mutations of amino acids in the pore-lining region of TASK-3 channels were introduced using site-directed mutagenesis., Results: Doxapram was an equipotent inhibitor of human TASK-1 and TASK-3 channels, compared with mouse channel variants, where it was more selective for TASK-1 and heterodimers of TASK-1 and TASK-3. The effect of doxapram could be attenuated by either the removal of the C-terminus of human TASK-3 channels or mutations of particular hydrophobic residues in the pore-lining region. These mutations, however, did not alter the effect of a known extracellular inhibitor of TASK-3, zinc. The positive enantiomer of doxapram, GAL-054, was a more potent antagonist of TASK channels, than doxapram, whereas the negative enantiomer, GAL-053, had little inhibitory effect., Conclusion: These data show that in contrast to rodent channels, doxapram is a potent inhibitor of both TASK-1 and TASK-3 human channels, providing further understanding of the pharmacological profile of doxapram in humans and informing the development of new therapeutic agents., (© 2019 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

The propagation of electrical impulses along axons is highly accelerated by the myelin sheath and produces saltating or "jumping" action potentials across internodes, from one node of Ranvier to the next. The underlying electrical circuit, as well as the existence and role of submyelin conduction in saltatory conduction remain, however, elusive. Here, we made patch-clamp and high-speed voltage-calibrated optical recordings of potentials across the nodal and internodal axolemma of myelinated neocortical pyramidal axons combined with electron microscopy and experimentally constrained cable modeling. Our results reveal a nanoscale yet conductive periaxonal space, incompletely sealed at the paranodes, which separates the potentials across the low-capacitance myelin sheath and internodal axolemma. The emerging double-cable model reproduces the recorded evolution of voltage waveforms across nodes and internodes, including rapid nodal potentials traveling in advance of attenuated waves in the internodal axolemma, revealing a mechanism for saltation across time and space., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Voltage-gated sodium channel Na v 1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of Na V 1.5 at 3.2-3.5 Å resolution. Na V 1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the Na V β subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na + ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Na v 1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The monoterpenoid terpinen-4-ol (4TERP) is known to inhibit cell excitability, has low toxicity and important pharmacological activities, which are likely related to neural excitability, such as anti-inflammatory, antiepileptic and antinociceptive effects. However, the pharmacological characteristics and mechanisms underlying the effects of 4TERP on blockade of neural action potential are not completely elucidated. Since Na + current (I Na ) through voltage-dependent Na + channels (Na V ) is a major mechanism for excitability, the present study investigated the pharmacological characteristics and mechanisms of the action of 4TERP on I Na through Na V . For this aim, dissociated small neurons of dorsal root ganglia of adult rats were used for whole cell patch-clamp recordings. 4TERP concentration-dependently inhibits I Na (IC 50 0.8 ± 0.3 mM; pharmacological efficacy 42.89 ± 5.54%). 4TERP interfered with I Na through a mechanism with various components, which includes predominantly channel pore block and sensitivity to frequency of use. In presence of 4TERP (3 mM), decreasing stimulation from 5 Hz to very low frequency (75 s of quiescence previously to stimulation) induced I Na decrease to 65.17 ± 5.86% of control. 4TERP also altered (left shift) voltage sensitivity of the steady state activation of Na V . Data are discussed aiming to interpret the importance of blockade of I Na through Na V as participant of 4TERP-induced inhibition of membrane excitability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The polygenic background of selectively bred diabetes models mimics the etiology of type 2 diabetes. So far, three different rodent models (Goto-Kakizaki rats, Nagoya-Shibata-Yasuda mice, and Oikawa-Nagao mice) have been established in the diabetes research field by continuous selective breeding for glucose tolerance from outbred rodent stocks. The origin of hyperglycemia in these rodents is mainly insulin secretion deficiency from the pancreatic β-cells and mild insulin resistance in insulin target organs. In this chapter, we summarize backgrounds and phenotypes of these rodent models to highlight their importance in diabetes research. Then, we introduce experimental methodologies to evaluate β-cell exocytosis as a putative common defect observed in these rodent models.

A detailed description of the electrophysiological features of cardiomyocytes in the various contractile chambers of the vertebrate heart is essential to understand the evolution of cardiac electrical activity, yet very little is known about reptiles. The present study characterizes major ionic currents (I Na , I CaL , I Kr , I K1 and I KACh ) and action potential (AP) configuration in cardiomyocytes from the ventricle, the right atrium and the sinus venosus (SV) of Burmese pythons (Python molurus) using sharp microelectrode and patch clamp recordings. Special attention was given to SV, since it consists of myocardial cells and appears to contribute to right atrial filling in snakes. We demonstrate that most of the SV in pythons has a stable resting potential of - 82.3 ± 2.6 mV (n = 9) and lacks pacemaker activity. AP duration at 50% repolarization was similar in cells from SV and atria (350.2 ± 8.7 and 330.4 ± 17.2 ms, respectively; n = 7), but shorter than ventricular APs (557.6 ± 19.2 ms, n = 5) at 30 °C. The densities of ionic currents, however, differed substantially between atrial and SV cells, where the latter had much lower densities of I Na , I CaL and I Kr than atrial and ventricular myocytes. I K1 in ventricle was ninefold greater than in atrial cells and 23-fold greater than in myocytes from SV. However, I KACh was absent in ventricular cells, while it was equally large in atrial and SV myocytes. Consistent with this observation, APs of atrium and SV, but not ventricle, were greatly shortened upon addition of acetylcholine (10 -6  M). Thus, snake SV, right atrium and ventricle have distinct patterns of ionic currents, but the resulting electrical activity is similar in atrium and SV.

The Slack (KCNT1) gene encodes sodium-activated potassium channels that are abundantly expressed in the central nervous system. Human mutations alter the function of Slack channels, resulting in epilepsy and intellectual disability. Most of the disease-causing mutations are located in the extended cytoplasmic C-terminus of Slack channels and result in increased Slack current. Previous experiments have shown that the C-terminus of Slack channels binds a number of cytoplasmic signaling proteins. One of these is Phactr1, an actin-binding protein that recruits protein phosphatase 1 (PP1) to certain phosphoprotein substrates. Using co-immunoprecipitation, we found that Phactr1 is required to link the channels to actin. Using patch clamp recordings, we found that co-expression of Phactr1 with wild-type Slack channels reduces the current amplitude but has no effect on Slack channels in which a conserved PKC phosphorylation site (S407) that regulates the current amplitude has been mutated. Furthermore, a Phactr1 mutant that disrupts the binding of PP1 but not that of actin fails to alter Slack currents. Our data suggest that Phactr1 regulates the Slack by linking PP1 to the channel. Targeting Slack-Phactr1 interactions may therefore be helpful in developing the novel therapies for brain disorders associated with the malfunction of Slack channels., (© 2019 Federation of American Societies for Experimental Biology.)

P2X receptors are a structurally and functionally distinctive family of ligand-gated ion channels that play important roles in mediating extracellular adenosine 5'-triphosphate (ATP) signaling in diverse physiological and pathophysiological processes. For several decades, the "manual" patch-clamp technique was regarded as the gold standard assay for investigating ion channel properties. More recently, breakthroughs in the development of automated patch-clamp technologies are enabling the study of ion channels, with much greater throughput capacities. These automated platforms, of which there are many, generate consistent, reliable, high-fidelity data. This chapter demonstrates the versatility of one of these technologies for ligand-gated ion channels, with a particular emphasis on protocols that address some of the issues of receptor desensitization that are commonly associated with P2X receptor-mediated currents.

Axonal damage can cause a loss of neural control of target peripheral muscles and other organs. The hallmark of complete recovery from severe axonal injury is a successful return of function. To assay the degree of functional loss or recovery from injury, a measurement of electrical communication at the nerve-target junction can be used. Drosophila larval neuromuscular junction (NMJ) provides a genetically tractable and easily accessible model to measure the electrophysiological properties of the synapse. To study the functional consequences of injuries to the peripheral nerve, we describe the procedure to measure the spontaneous and evoked response to neurotransmitter release at the NMJ.

Gap junction-mediated electrical coupling between retinal photoreceptors is an important determinant of photoreceptor function. Yet, quantitative measurements of the junctional conductance between coupled photoreceptors are required to fully assess the effects of coupling on visual performance. Such measurements have been obtained in salamander and other lower vertebrate retinas but are difficult to acquire in mammalian retinas, in part because of the much smaller size of photoreceptors in mammals. Here, we describe in detail a dual whole-cell patch-clamp technique we recently developed to measure the junctional conductance between photoreceptor pairs in the mouse retina. With this method, electrical coupling strength between mouse photoreceptors can be estimated with high accuracy and its impact on retinal processing of visual information further evaluated.

Xenopus oocytes serve as a standard heterologous expression system for the study of various ligand-gated ion channels including ATP P2X receptors. Here we describe the whole-cell two-electrode voltage clamp and biotinylation/Western blotting techniques to investigate the functional properties and surface trafficking from P2X-expressing oocytes.

P2X receptors (P2XRs) are ligand-gated ion channels gated by extracellular adenosine 5'-triphosphate (ATP) and play a critical role in mediating ATP-induced purinergic signaling in physiological and pathological processes. Heterologous expression of P2XR in human embryonic kidney 293 (HEK293) cells and measurement of P2XR-mediated currents using patch-clamp recording technique have been widely used to study the biophysical and pharmacological properties of these receptors. Combination of electrophysiology with site-directed mutagenesis and structural information has shed light on the molecular basis for receptor activation and mechanisms of actions by receptor antagonists and modulators. It is anticipated that such methodologies will continue helping us to provide more mechanistic understanding of P2XRs and to test novel receptor antagonists and allosteric modulators for therapeutical purposes. In this chapter, we describe protocols of transiently or stably expressing the P2XR in HEK293 cells and measuring P2XR-mediated currents by using whole-cell recording.

Investigating ion channels in their native cell type is important when striving to understand their regulation and function, but this comes with added complexities due to the plethora of channels and receptors present. Details of recording ATP-gated ion channels in macrophages are presented together with information on how to prepare the primary cells for electrophysiological analysis.

Plants possess numerous ion channels that respond to a range of stimuli, including small molecules, transmembrane voltage, and mechanical force. Many in the latter category, known as mechanosensitive (MS) ion channels, open directly in response to increases in lateral membrane tension. One of the most effective techniques for characterizing ion channel properties is patch-clamp electrophysiology, in which the current through a section of membrane containing ion channels is measured. For MS channels, this technique enables the measurement of key channel properties such as tension sensitivity, conductance, and ion selectivity. These characteristics, along with the phenotypes of genetic mutants, can help reveal the physiological roles of a particular MS channel. In this protocol, we provide detailed instructions on how to study MS ion channels using single-channel patch-clamp electrophysiology in giant E. coli spheroplasts. We first present an optimized method for preparing giant spheroplasts, then describe how to measure MS channel activity using patch-clamp electrophysiology and analyze the resulting data. We also provide recommended equipment lists, setup schematics, and useful conventions., (© 2020 Elsevier Inc. All rights reserved.)

The hERG potassium channel influences ventricular action potential duration. Extracellular acidosis occurs in pathological states including cardiac ischaemia. It reduces the amplitude of hERG current and speeds up deactivation, which can alter cardiac excitability. This study aimed to identify the site of action by which extracellular protons regulate the amplitude of macroscopic hERG current. Recordings of macroscopic and single hERG1a and 1b channel activity, mutagenesis, and the recent cryoEM structure for hERG were employed. Single hERG1a and 1b channels displayed open times that decreased with membrane depolarization, suggestive of a blocking mechanism that senses approximately 20% of the membrane electric field. This mechanism was sensitive to pH; extracellular acidosis reduced both hERG1a and1b channel open time and conductance. The effects of acidosis on macroscopic current amplitude and deactivation displayed different sensitivities to protons. Point mutation of a pair of residues (E575/H578) in the pore turret abolished the acidosis-induced decrease of current amplitude, without affecting the change in current deactivation. In single hERG1a channel recordings, the conductance of the double-mutant channel was unaffected by extracellular acidosis. These findings identify residues in the outer turret of the hERG channel that act as a proton sensor to regulate open time and channel conductance.

Aims: To explore the atrial electrical remodeling and the susceptibility of atrial fibrillation (AF) in diabetic rats., Materials and Methods: Zucker diabetic fatty (ZDF) rats were chosen as diabetic animal model, and age-matched non-diabetic littermate Zucker lean (ZL) rats as control. AF susceptibility was determined by electrophysiological examination. The current density of I to , I Kur and I Ca-L were detected by whole-cell patch-clamp technique, and ion channel protein expression in atrial tissue and HL-1 cells treated with advanced glycation end products (AGE) was analyzed by western blotting., Key Findings: Diabetic rats had significantly enlarged left atria and evenly thickened ventricular walls, hypertrophied cells and interstitial fibrosis in atrial myocardium, increased AF susceptibility, and prolonged AF duration after atrial burst stimulation. Compared with atrial myocytes isolated from ZL controls, atrial myocytes isolated from ZDF rats had prolonged action potential duration, decreased absolute value of resting membrane potential level and current densities of I to , I Kur and I Ca-L . The ion channel protein (Kv4.3, Kv1.5 and Cav1.2) expression in atrium tissue of ZDF rats and HL-1 cells treated with high concentration AGE were significantly down-regulated, compared with controls., Significance: The atrial electrical remodeling induced by hyperglycemia contributed to the increased AF susceptibility in diabetic rats., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The outer hair cell (OHC) of the organ of Corti underlies a process that enhances hearing, termed cochlear amplification. The cell possesses a unique voltage-sensing protein, prestin, that changes conformation to cause cell length changes, a process termed electromotility (eM). The prestin voltage sensor generates a capacitance that is both voltage- and frequency-dependent, peaking at a characteristic membrane voltage (V h ), which can be greater than the linear capacitance of the OHC. Accordingly, the OHC membrane time constant depends upon resting potential and the frequency of AC stimulation. The confounding influence of this multifarious time constant on eM frequency response has never been addressed. After correcting for this influence on the whole-cell voltage clamp time constant, we find that both guinea pig and mouse OHC eM is low pass, substantially attenuating in magnitude within the frequency bandwidth of human speech. The frequency response is slowest at V h , with a cut-off, approximated by single Lorentzian fits within that bandwidth, near 1.5 kHz for the guinea pig OHC and near 4.3 kHz for the mouse OHC, each increasing in a U-shaped manner as holding voltage deviates from V h Nonlinear capacitance (NLC) measurements follow this pattern, with cut-offs about double that for eM. Macro-patch experiments on OHC lateral membranes, where voltage delivery has high fidelity, confirms low pass roll-off for NLC. The U-shaped voltage dependence of the eM roll-off frequency is consistent with prestin's voltage-dependent transition rates. Modeling indicates that the disparity in frequency cut-offs between eM and NLC may be attributed to viscoelastic coupling between prestin's molecular conformations and nanoscale movements of the cell, possibly via the cytoskeleton, indicating that eM is limited by the OHC's internal environment, as well as the external environment. Our data suggest that the influence of OHC eM on cochlear amplification at higher frequencies needs reassessment., (© 2019 Santos-Sacchi et al.)

The 5-hydroxytryptamine (5-HT) type 3 receptor is a member of the cysteine (Cys)-loop receptor super family of ligand-gated ion channels in the nervous system and is a clinical target in a range of diseases. The 5-HT 3 receptor mediates fast serotonergic neurotransmission by undergoing a series of conformational changes initiated by ligand binding that lead to the rapid opening of an intrinsic cation-selective channel. However, despite the availability of high-resolution structures of a mouse 5-HT 3 receptor, many important aspects of the mechanistic basis of 5-HT 3 receptor function and modulation by drugs remain poorly understood. In particular, there is little direct evidence for the specific conformational changes predicted to occur during ligand-gated channel activation and desensitization. In the present study, we used voltage-clamp fluorometry (VCF) to measure conformational changes in regions surrounding the orthosteric binding site of the human 5-HT 3A (h5-HT 3A ) receptor during binding of 5-HT and different classes of 5-HT 3 receptor ligands. VCF utilizes parallel measurements of receptor currents with photon emission from fluorescent reporter groups covalently attached to specific positions in the receptor structure. Reporter groups that are highly sensitive to the local molecular environment can, in real time, report conformational changes as changes in fluorescence that can be correlated with changes in receptor currents reporting the functional states of the channel. Within the loop C, D, and E regions that surround the orthosteric binding site in the h5-HT 3A receptor, we identify positions that are amenable to tagging with an environmentally sensitive reporter group that reports robust fluorescence changes upon 5-HT binding and receptor activation. We use these reporter positions to characterize the effect of ligand binding on the local structure of the orthosteric binding site by agonists, competitive antagonists, and allosterically acting channel activators. We observed that loop C appears to show distinct fluorescence changes for ligands of the same class, while loop D reports similar fluorescence changes for all ligands binding at the orthosteric site. In contrast, the loop E reporter position shows distinct changes for agonists, antagonists, and allosteric compounds, suggesting the conformational changes in this region are specific to ligand function. Interpretation of these results within the framework of current models of 5-HT 3 and Cys-loop mechanisms are used to expand the understanding of how ligand binding in Cys-loop receptors relates to channel gating. SIGNIFICANCE STATEMENT: The 5-HT 3 receptor is an important ligand-gated ion channel and drug target in the central and peripheral nervous system. Determining how ligand binding induced conformational changes in the receptor is central for understanding the structural mechanisms underlying 5-HT 3 receptor function. Here, we employ voltage-gated fluorometry to characterize conformational changes in the extracellular domain of the human 5-HT 3 receptor to identify intrareceptor motions during binding of a range of 5-HT 3 receptor agonists and antagonists., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The hippocampus is involved in key neuronal circuits that underlie cognition, memory, and anxiety, and it is increasingly recognized as a vulnerable structure that contributes to the pathogenesis of HIV-associated neurocognitive disorder (HAND). However, the mechanisms responsible for hippocampal dysfunction in neuroHIV remain unknown. The present study used HIV transgenic (Tg) rats and patch-clamp electrophysiological techniques to study the effects of the chronic low-level expression of HIV proteins on hippocampal CA1 pyramidal neurons. The dorsal and ventral areas of the hippocampus are involved in different neurocircuits and thus were evaluated separately. We found a significant decrease in the intrinsic excitability of CA1 neurons in the dorsal hippocampus in HIV Tg rats by comparing neuronal spiking induced by current step injections and by dynamic clamp to simulate neuronal spiking activity. The decrease in excitability in the dorsal hippocampus was accompanied by a higher rate of excitatory postsynaptic currents (EPSCs), whereas CA1 pyramidal neurons in the ventral hippocampus in HIV Tg rats had higher EPSC amplitudes. We also observed a reduction of hyperpolarization-activated nonspecific cationic current (I h ) in both the dorsal and ventral hippocampus. Neurotoxic HIV proteins have been shown to increase neuronal excitation. The lower excitability of CA1 pyramidal neurons that was observed herein may represent maladaptive homeostatic plasticity that seeks to stabilize baseline neuronal firing activity but may disrupt neural network function and contribute to HIV-associated neuropsychological disorders, such as HAND and depression., (Copyright © 2019. Published by Elsevier B.V.)

We describe the first extensive study of voltage-clamp current responses of cone photoreceptors in unlabeled, dark-adapted mouse retina using only the position and appearance of cone somata as a guide. Identification was confirmed from morphology after dye filling. Photocurrents recorded from wild-type mouse cones were biphasic with a fast cone component and a slower rod component. The rod component could be eliminated with dim background light and was not present in mouse lines lacking the rod transducin-α subunit ( Gnat1 -/- ) or connexin 36 ( Cx36 -/- ). Cones from Gnat1 -/- or Cx36 -/- mice had resting membrane potentials between -45 and -55 mV, peak photocurrents of 20-25 picoamps (pA) at a membrane potential V m = -50 mV, sensitivities 60-70 times smaller than rods, and a total membrane capacitance two to four times greater than rods. The rate of activation (amplification constant) was largely independent of the brightness of the flash and was 1-2 s -2 , less than half that of rods. The role of Ca 2+ -dependent transduction modulation was investigated by recording from cones in mice lacking rod transducin (Gnat1), recoverin, and/or the guanylyl-cyclase-activating proteins (GCAPs). In confirmation of previous results, responses of Gnat1 -/- ; Gcaps -/- cones and triple-mutant Gnat1 -/- ; Gcaps -/- ; Rv -/- cones recovered more slowly both to light flashes and steps and were more sensitive than cones expressing the GCAPs. Cones from all four mouse lines showed significant recovery and escaped saturation even in bright background light. This recovery occurred too rapidly to be caused by pigment bleaching or metaII decay and appears to reflect some modulation of response inactivation in addition to those produced by recoverin and the GCAPs. Our experiments now make possible a more detailed understanding of the cellular physiology of mammalian cone photoreceptors and the role of conductances in the inner and outer segment in producing cone light responses., (© 2019 Fain et al.)

Key Points: Imbalances in the activity of the D1-expressing direct pathway and D2-expressing indirect pathway striatal projection neurons (SPNs) are thought to contribute to many basal ganglia disorders, including early-onset neurodevelopmental disorders such as obsessive-compulsive disorder, attention deficit hyperactivity disorder and Tourette's syndrome. This study provides the first detailed quantitative investigation of development of D1 and D2 SPNs, including their cellular properties and connectivity within neural circuits, during the first postnatal weeks. This period is highly dynamic with many properties changing, but it is possible to make three main observations: many aspects of D1 and D2 SPNs progressively mature in parallel; there are notable exceptions when they diverge; and many of the defining properties of mature striatal SPNs and circuits are already established by the first and second postnatal weeks, suggesting guidance through intrinsic developmental programmes. These findings provide an experimental framework for future studies of striatal development in both health and disease., Abstract: Many basal ganglia neurodevelopmental disorders are thought to result from imbalances in the activity of the D1-expressing direct pathway and D2-expressing indirect pathway striatal projection neurons (SPNs). Insight into these disorders is reliant on our understanding of normal D1 and D2 SPN development. Here we provide the first detailed study and quantification of the striatal cellular and circuit changes occurring for both D1 and D2 SPNs in the first postnatal weeks using in vitro whole-cell patch-clamp electrophysiology. Characterization of their intrinsic electrophysiological and morphological properties, the excitatory long-range inputs coming from cortex and thalamus, as well their local gap junction and inhibitory synaptic connections reveals this period to be highly dynamic with numerous properties changing. However it is possible to make three main observations. Firstly, many aspects of SPNs mature in parallel, including intrinsic membrane properties, increases in dendritic arbours and spine densities, general synaptic inputs and expression of specific glutamate receptors. Secondly, there are notable exceptions, including a transient stronger thalamic innervation of D2 SPNs and stronger cortical NMDA receptor-mediated inputs to D1 SPNs, both in the second postnatal week. Thirdly, many of the defining properties of mature D1 and D2 SPNs and striatal circuits are already established by the first and second postnatal weeks, including different electrophysiological properties as well as biased local inhibitory connections between SPNs, suggesting this is guided through intrinsic developmental programmes. Together these findings provide an experimental framework for future studies of D1 and D2 SPN development in health and disease., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

It has been shown that dystrobrevin-binding protein 1 gene that encodes the protein dysbindin-1 is associated with risk for cognitive deficits, and studies have shown decreases in glutamate and correlated decreases in dysbindin-1 protein in the prefrontal cortex (PFC) and hippocampus of post-mortem tissue from schizophrenia patients. The PFC and the hippocampus have been shown to play a fundamental role in cognition, and studies in dysbindin-1 null mice have shown alterations in NMDAR located in pyramidal neurons as well as perturbation in LTP and cognitive deficits. The balance between excitatory and inhibitory transmission is crucial for normal cognitive functions; however, there is a dearth of information regarding the effects of loss of dysbindin-1 in GABAergic transmission. Using in vitro whole-cell clamp recordings, Western blots, and immunohistochemistry, we report here that dysbindin-1-deficient mice exhibit a significant decrease in the frequency of sIPSCs and in the amplitude of mIPSCs and significant decreases in PV staining and protein level. These results suggest that loss of dysbindin-1 affects GABAergic transmission at pre- and postsynaptic level and decreases parvalbumin markers.

Nociceptive trigeminal afferents innervating craniofacial area, eg, facial skin and cranial meninges, project to a broad region in the medullary and upper cervical dorsal horn designated as the trigeminocervical complex. Lamina I neurons in the trigeminocervical complex integrate and relay peripheral inputs, thus playing a key role in both cranial nociception and primary headache syndromes. Because of the technically challenging nature of recording, the long-range trigeminal afferent inputs to the medullary and cervical lamina I neurons were not intensively studied so far. Therefore, we have developed an ex vivo brainstem-cervical cord preparation with attached trigeminal nerve for the visually guided whole-cell recordings from the medullary and cervical lamina I neurons. Two-thirds of recorded neurons generated intrinsic rhythmic discharges. The stimulation of the trigeminal nerve produced a complex effect; it interrupted the rhythmic discharge for hundreds of milliseconds but, if the neuron was silenced by a hyperpolarizing current injection, could elicit a discharge. The monosynaptic inputs from the trigeminal Aδ, high-threshold Aδ, low-threshold C, and C afferents were recorded in the medullary neurons, as well as in the cervical neurons located in the segments C1 to C2 and, to a lesser degree, in C3 to C4. This pattern of supply was consistent with our labelling experiments showing extensive cervical projections of trigeminal afferents. Excitatory inputs were mediated, although not exclusively, through AMPA/kainate and NMDA receptors, whereas inhibitory inputs through both GABA and glycine receptors. In conclusion, the trigeminocervical lamina I neurons receive a complex pattern of long-range monosynaptic and polysynaptic inputs from a variety of the trigeminal nociceptive afferents.

Charge selectivity forms the basis of cellular excitation or inhibition by Cys-loop ligand-gated ion channels (LGICs), and is essential for physiological receptor function. There are no reports of naturally occurring mutations in LGICs associated with the conversion of charge selectivity. Here, we report on a CHRNA1 mutation (α1Leu251Arg) in a patient with congenital myasthenic syndrome associated with transformation of the muscle acetylcholine receptor (AChR) into an inhibitory channel. Performing patch-clamp experiments, the AChR was found to be converted into chloride conductance at positive potentials, whereas whole-cell currents at negative potentials, although markedly reduced, were still carried by sodium. Umbrella sampling molecular dynamics simulations revealed constriction of the channel pore radius to 2.4 Å as a result of the mutation, which required partial desolvation of the ions in order to permeate the pore. Ion desolvation was associated with an energetic penalty that was compensated for by the favorable electrostatic interaction of the positively charged arginines with chloride. These findings reveal a mechanism for the transformation of the muscle AChR into an inhibitory channel in a clinical context., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Key advances in single particle cryo-EM methods in the past decade have ushered in a resolution revolution in modern biology. The structures of many ion channels and transporters that were previously recalcitrant to crystallography have now been solved. Yet, despite having atomistic models of many complexes, some in multiple conformations, it has been challenging to glean mechanistic insight from these structures. To some extent this reflects our inability to unambiguously assign a given structure to a particular physiological state. One approach that may allow us to bridge this gap between structure and function is voltage clamp fluorometry (VCF). Using this technique, dynamic conformational changes can be measured while simultaneously monitoring the functional state of the channel or transporter. Many of the important papers that have used VCF to probe the gating mechanisms of channels and transporters have been published in the Journal of General Physiology In this review, we provide an overview of the development of VCF and discuss some of the key problems that have been addressed using this approach. We end with a brief discussion of the outlook for this technique in the era of high-resolution structures., (© 2019 Cowgill and Chanda.)

Neural network processing is usually studied using the spike times of many extracellularly recorded neurons. Elucidating the cellular-synaptic mechanisms underlying these firing patterns requires identifying and controlling single cells and assessing their inputs. Single cell glass electrode techniques (intracellular, patch and juxtacellular) are well suited to filling this gap, in terms of physiology, cell identity and behavior. However, they are typically limited to in vitro and immobilized in vivo experiments, primarily due to the necessity for mechanical stability and steep learning curves. Several approaches have been recently developed to extend these technologies to freely moving animals. Here we summarize the advantages and results for different methods of single neuron glass recordings in vivo. We further review three approaches used to date for single cell recording in freely moving animals: static anchor systems, manual mechanic drives and motorized drives. Finally, we highlight new technologies capable of expanding the utility of single neuron recording in freely moving animals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Electrophysiology is the study of neural activity in the form of local field potentials, current flow through ion channels, calcium spikes, back propagating action potentials and somatic action potentials, all measurable on a millisecond timescale. Despite great progress in imaging technologies and sensor proteins, none of the currently available tools allow imaging of neural activity on a millisecond timescale and beyond the first few hundreds of microns inside the brain. The patch clamp technique has been an invaluable tool since its inception several decades ago and has generated a wealth of knowledge about the nature of voltage- and ligand-gated ion channels, sub-threshold and supra-threshold activity, and characteristics of action potentials related to higher order functions. Many techniques that evolve to be standardized tools in the biological sciences go through a period of transformation in which they become, at least to some degree, automated, in order to improve reproducibility, throughput and standardization. The patch clamp technique is currently undergoing this transition, and in this review, we will discuss various aspects of this transition, covering advances in automated patch clamp technology both in vitro and in vivo., (Copyright © 2019. Published by Elsevier B.V.)

Whole-cell patch clamp recordings demonstrated that in the dentate gyrus (DG) as well as in the CA3 area of mouse hippocampal slices the prototypic P2X7 receptor (R) agonist dibenzoyl-ATP (Bz-ATP) induced inward current responses both in neurons and astrocytes. Whereas the selective P2X7R antagonist A438079 strongly inhibited both neuronal and astrocytic currents, a combination of ionotropic glutamate receptor (CNQX, AP-5) and GABA A -R (gabazine) antagonists depressed the Bz-ATP-induced current responses in the DG (granule cells) and CA3 neurons only. It was concluded that Bz-ATP activated astrocytic P2X7Rs and thereby released glutamate and GABA to stimulate nearby neurons. The residual A438079-resistant current response of astrocytes was suggested to be due to the stimulation of P2XRs of the non-P2X7-type. Further, we searched for presynaptic P2X7Rs at the axon terminals of DG and CA3 pyramidal neurons innervating CA3 and CA1 cells, respectively. Bz-ATP potentiated the frequency of spontaneous postsynaptic currents (sPSCs) in CA1 but not CA3 pyramidal cells. However, the Bz-ATP effect in CA1 cells was inhibited by gabazine or the astrocytic toxin fluorocitrate suggesting stimulation of P2X7Rs at stratum radiatum astrocytes located near to interneurons and synapsing onto CA1 neurons. Our data suggest that functional P2X7Rs are missing at neurons in the tri-synaptic network of the rodent hippocampus, but are present at nearby astrocytes indirectly regulating network activity., (Copyright © 2018 Elsevier Inc. All rights reserved.)