Patch-clamp Research Articles

Distinct effects of obesity and diabetes on the action potential waveform and inward currents in rat ventricular myocytes.

Obesity is a significant global health challenge, increasing the risk of developing type 2 diabetes mellitus (T2DM) and cardiovascular disease. Research indicates that obese individuals, regardless of their diabetic status, have an increased risk of cardiovascular complications. Studies suggest that these patients experience impaired electrical conduction in the heart, although the underlying cause-whether due to obesity-induced fat toxicity or diabetes-related factors-remains uncertain. This study investigated ventricular action potential parameters, as well as sodium (INa) and calcium (ICa, L) currents, in Zucker fatty (ZF) rats and Zucker diabetic fatty (ZDF) rats, which serve as models for obesity and T2DM, respectively. &#160;Ventricular myocytes were isolated from 25-30-week-old Zucker rats. Resting and action potentials were recorded using a β-Escin perforated patch clamp, while INa and ICa, L were assessed with whole-cell patch clamp methods. ZF rats exhibited higher excitability and faster upstroke velocity with greater INa density, whereas ZDF rats showed decreased INa and slower action potential upstroke. No differences in ICa, L density or voltage sensitivity were found among the groups.</p> In summary, obesity, with or without accompanying T2DM, distinctly impacts the action potential waveform, INa density, and excitability of ventricular myocytes in this rat model of T2DM.

Quetiapine competitively inhibits 5-HT3 receptor-mediated currents in NCB20 neuroblastoma cells.

The 5-hydroxytryptamine type3 (5-HT3) receptor, a ligand-gated ion channel, plays a critical role in synaptic transmission. It has been implicated in various neuropsychiatric disorders. This study aimed to elucidate the mechanism by which quetiapine, an atypical antipsychotic, could inhibit 5-HT3 receptor-mediated currents in NCB20 neuroblastoma cells. Whole-cell patch-clamp recordings were used to study effects of quetiapine on receptor ion channel kinetics and its competitive antagonism. Co-application of quetiapine shifted 5-HT concentration-response curve rightward, significantly increasing the EC50 without altering the maximal response (Emax), suggesting a competitive inhibition. Quetiapine's IC50 varied with 5-HT concentration and treatment condition. The IC50 value of quetiapine was 0.58 μM with 3 μM 5-HT and 25.23 μM with 10 μM 5-HT, indicating an inverse relationship between quetiapine efficacy and agonist concentration. Pretreatment of quetiapine significantly enhanced its inhibitory potency, reducing its IC50 from 25.23 μM to 0.20 μM. Interaction kinetics experiments revealed an IC50 of 5.17 μM for an open state of the 5-HT3 receptor, suggesting weaker affinity during receptor activation. Quetiapine also accelerated receptor deactivation and desensitization, suggesting that it could stabilize the receptor in non-conducting states. Additionally, quetiapine significantly prolonged recovery from desensitization without affecting recovery from deactivation, demonstrating its selective impact on receptor kinetics. Inhibition of the 5-HT3 receptor by quetiapine was voltage-independent, and quetiapine exhibited no usedependency, further supporting its role as a competitive antagonist. These findings provide insights into inhibitory mechanism of quetiapine on 5-HT3 receptor and suggest its potential therapeutic implications for modulating serotonergic pathways in neuropsychiatric disorders.

Slick potassium channels limit TRPM3-mediated activation of sensory neurons

Heat sensation is mediated by specialized heat-sensitive neurons in the somatosensory system that innervates the skin. Previous studies revealed that noxious heat sensation is controlled by the sodium (Na+)-activated potassium (K+) channel Slick (Kcnt2), which is highly expressed in nociceptive Aδ-fibers. However, the mechanism by which Slick modulates heat sensation is poorly understood. Here, we generated mice lacking Slick conditionally in sensory neurons expressing Nav1.8 (SNS-Slick−/− mice). In SNS-Slick−/− mice, the latency to express any nocifensive behavior was reduced in the hot plate and tail immersion tests. In situ hybridization experiments revealed Slick was highly co-expressed with the essential heat sensor, transient receptor potential (TRP) melastatin (TRPM) 3, but not with TRP vanilloid 1, TRP ankyrin 1, or TRPM2 in sensory neurons. Notably, SNS-Slick−/− mice exhibited increased nocifensive behaviors following intraplantar injection of the TRPM3 activator pregnenolone sulfate. Patch-clamp recordings detected increased Na+-dependent outward K+ current (IK) after TRPM3 activation in sensory neurons, which showed no prominent IK after the replacement of NaCl with choline chloride. Thus, our study suggests that Slick limits TRPM3-mediated activation of sensory neurons, thereby inhibiting noxious heat sensing.

Chemogenetic Inhibition of Prefrontal Cortex Ameliorates Autism-Like Social Deficits and Absence-Like Seizures in a Gene-Trap Ash1l Haploinsufficiency Mouse Model

Background: ASH1L (absent, small, or homeotic-like 1), a histone methyltransferase, has been identified as a high-risk gene for autism spectrum disorder (ASD). We previously showed that postnatal Ash1l severe deficiency in the prefrontal cortex (PFC) of male and female mice caused seizures. However, the synaptic mechanisms underlying autism-like social deficits and seizures need to be elucidated. Objective: The goal of this study is to characterize the behavioral deficits and reveal the synaptic mechanisms in an Ash1l haploinsufficiency mouse model using a targeted gene-trap knockout (gtKO) strategy. Method: A series of behavioral tests were used to examine behavioral deficits. Electrophysiological and chemogenetic approaches were used to examine and manipulate the excitability of pyramidal neurons in the PFC of Ash1l+/GT mice. Results: Ash1l+/GT mice displayed social deficits, increased self-grooming, and cognitive impairments. Epileptiform discharges were found on electroencephalograms (EEGs) of Ash1l+/GT mice, indicating absence-like seizures. Ash1l haploinsufficiency increased the susceptibility for convulsive seizures when Ash1l+/GT mice were challenged by pentylenetetrazole (PTZ, a competitive GABAA receptor antagonist). Whole-cell patch-clamp recordings showed that Ash1l haploinsufficiency increased the excitability of pyramidal neurons in the PFC by altering intrinsic neuronal properties, enhancing glutamatergic synaptic transmission, and diminishing GABAergic synaptic inhibition. Chemogenetic inhibition of pyramidal neurons in the PFC of Ash1l+/GT mice ameliorated autism-like social deficits and abolished absence-like seizures. Conclusions: We demonstrated that increased neural activity in the PFC contributed to the autism-like social deficits and absence-like seizures in Ash1l+/GT mice, which provides novel insights into the therapeutic strategies for patients with ASH1L-associated ASD and epilepsy.

Pimozide Inhibits Type II but Not Type I Hair Cells in Chicken Embryo and Adult Mouse Vestibular Organs

Background: Pimozide is a conventional antipsychotic drug of the diphenylbutylpiperidine class, widely used for treating schizophrenia and delusional disorders and for managing motor and phonic tics in Tourette’s syndrome. Pimozide is known to block dopaminergic D2 receptors and various types of voltage-gated ion channels. Among its side effects, dizziness and imbalance are the most frequently observed, which may imply an effect of the drug on the vestibular sensory receptors, the hair cells. Amniotes possess two classes of vestibular hair cells, named type I and type II hair cells, which differ in terms of signal processing and transmission. We previously reported that Pimozide [3 μM] significantly increased a delayed outward rectifying K+ current (IK,V). Methods and Results: In the present study, using the whole-cell patch-clamp technique we additionally show that Pimozide decreases the inward rectifying K+ current (IK,1) and the mixed Na+/K+ current (Ih) of chicken embryo type II hair cells, whereas it does not affect type I hair cells’ ionic currents. Since ion channels’ expression can vary depending on age and animal species, in the present study, we also tested Pimozide in adult mouse vestibular hair cells. We found that, like in the chicken embryo, Pimozide significantly increases IK,V and decreases IK,1 and Ih in type II hair cells. However, in the adult mouse, Pimozide also slightly increased the outward rectifying K+ current in type I hair cells. Conclusions: While providing a possible explanation for the vestibular side effects of Pimozide in humans, its inhibitory action on mammalian hair cells might be of interest for the local treatment of vestibular disorders characterized by altered vestibular input, like Ménière’s disease.

Inositol 1,4,5-Trisphosphate Receptor 1 Gain-of-Function Increases the Risk for Cardiac Arrhythmias in Mice and Humans.

Ca2+ mishandling in cardiac Purkinje cells is a well-known cause of cardiac arrhythmias. The Purkinje cell resident inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is believed to play an important role in Ca2+ handling, and ITPR1 gain-of-function (GOF) has been implicated in cardiac arrhythmias. However, nearly all known disease-associated ITPR1 variants are loss-of-function and are primarily linked to neurological disorders. Whether ITPR1 GOF has pathological consequences, such as cardiac arrhythmias, is unclear. This study aimed to identify human ITPR1 GOF variants and determine the impact of ITPR1 GOF on Ca2+ handling and arrhythmia susceptibility. There are a large number of rare ITPR1 missense variants reported in open data repositories. Based on their locations in the ITPR1 channel structure, we selected and characterized 33 human ITPR1 missense variants from open databases and identified 21 human ITPR1 GOF variants. We generated a mouse model carrying a human ITPR1 GOF variant, ITPR1-W1457G (W1447G in mice). We showed that the ITPR1-W1447G± and recently reported ITPR1-D2594K± GOF mutant mice were susceptible to stress-induced ventricular arrhythmias. Confocal Ca2+ and voltage imaging in situ in heart slices and Ca2+ imaging and patch-clamp recordings of isolated Purkinje cells showed that ITPR1-W1447G± and ITPR1-D2594K± variants increased the occurrence of stress-induced spontaneous Ca2+ release, delayed afterdepolarization, and triggered activity in Purkinje cells. To assess the potential role of ITPR1 variants in arrhythmia susceptibility in humans, we looked up a gene-based association study in the UK Biobank data set and identified 7 rare ITPR1 missense variants showing potential association with cardiac arrhythmias. Remarkably, in vitro functional characterization revealed that all these 7 ITPR1 variants resulted in GOF. Our studies in mice and humans reveal that enhanced function of ITPR1, a well-known movement disorder gene, increases the risk for cardiac arrhythmias.

Delta opioid receptor agonists activate PI3K-mTORC1 signaling in parvalbumin-positive interneurons in mouse infralimbic prefrontal cortex to exert acute antidepressant-lie effects.

The delta opioid receptor (DOP) is a promising target for novel antidepressants due to its potential for rapid action with minimal adverse effects; however, the functional mechanism underlying acute antidepressant actions remains elusive. We report that subcutaneous injection of the selective DOP agonist KNT-127 reduced immobility in the forced swimming test, and that this antidepressant-like response was reversed by intracerebroventricular injection of the selective mechanistic (or mammalian) target of rapamycin (mTOR) inhibitor rapamycin or the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002. KNT-127 also alleviated social avoidance and reduced sucrose consumption (anhedonia) among chronic vicarious social defeat stress model mice, which were similarly reversed by PI3K and mTOR inhibitors. In addition, KNT-127 increased phosphorylation levels of the mTOR signaling-related proteins Akt and p70S6 kinase in medial prefrontal cortex as revealed by immunoblotting. In the forced swimming test, a microinfusion of KNT-127 and another DOP agonist SNC80 in the infralimbic prefrontal cortex (IL-PFC) attenuated the immobility, which were blocked by rapamycin and LY294002. Perfusion of KNT-127 onto IL-PFC slices increased miniature excitatory postsynaptic current frequency and reduced miniature inhibitory postsynaptic current frequency in pyramidal neurons as measured by whole-cell patch-clamping, and both responses were reversed by rapamycin. Imaging of brain slices from transgenic mice with DOP-promoter-driven green fluorescent protein revealed that most DOPs were expressed in parvalbumin-positive interneurons in the IL-PFC. These findings suggest that DOP agonists exert antidepressant-like actions by suppressing GABA release from parvalbumin-positive interneurons via the PI3K-Akt-mTORC1-p70S6 kinase pathway, thereby enhancing IL-PFC pyramidal neuron excitation.

Sex-specific age-related changes in excitatory and inhibitory intra-cortical circuits in mouse primary auditory cortex.

A common impairment in aging is age-related hearing loss (presbycusis), which manifests as impaired spectrotemporal processing. Presbycusis can be caused by a dysfunction of the peripheral and central auditory system and these dysfunctions might differ between the sexes. To date, the circuit mechanisms in the central nervous system responsible for age-related auditory dysfunction remain mostly unknown. In the auditory cortex (ACtx), aging is accompanied by alteration in normal inhibitory (GABA) neurotransmission and changes in excitatory (NMDA and AMPA) synapses, but which circuits are affected has been unclear. Here we investigated how auditory cortical microcircuits change with age and if sex-dependent differences existed. We performed laser-scanning photostimulation (LSPS) combined with whole-cell patch clamp recordings from Layer (L) 2/3 cells in primary auditory cortex (A1) in young adult (2-3 months) and aged (older than 18 months) male and female CBA/CaJ mice which have normal peripheral hearing. We found that L2/3 cells in aged male animals display functional hypoconnectivity of inhibitory circuits originating from L2/3 and L4. Compared to cells from young adult mice, cells from aged male mice have weaker excitatory connections from L2/3. We also observed an increased diversity of excitatory and inhibitory inputs. These results suggest a sex-specific reduction and diversification in excitatory and inhibitory intralaminar cortical circuits in aged mice compared with young adult animals. We speculate that these unbalanced changes in cortical circuits contribute to the functional manifestations of age-related hearing loss in both males and females.Significance Statement A common impairment in aging is age-related hearing loss (presbycusis) which can be caused by a dysfunction of the peripheral and central auditory system. Impaired spectrotemporal processing in the auditory cortex is thought to underlie some of these impairments. We compare auditory cortex circuits in vitro in young adult (∼P60) and aged (∼P585) CBA mice. We find a sex-specific reduction in excitatory and inhibitory intralaminar cortical circuits in aged mice compared with young adult animals. We speculate that these unbalanced changes in cortical circuits underlie the differential functional manifestations of age-related hearing loss in both males and females.

Multiplexed Assays of Variant Effect and Automated Patch Clamping Improve KCNH2-LQTS Variant Classification and Cardiac Event Risk Stratification.

Long QT syndrome is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2. Variant classification is difficult, often because of lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of KCNH2 missense variant heterozygotes. We quantified cell-surface trafficking of 18 796 variants in KCNH2 using a multiplexed assay of variant effect (MAVE). We recorded KCNH2 current density for 533 variants by automated patch clamping. We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1458 patients with KCNH2 missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian long QT syndrome penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events. Variant MAVE trafficking scores and automated patch clamping peak tail currents were highly correlated (Spearman rank-order ρ=0.69; n=433). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian long QT syndrome penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and corrected QT interval (QTc); however, MAVE data became nonsignificant when peak tail current and penetrance estimates were also available. The area under the receiver operator characteristic curve for 20-year event outcomes based on patient-specific sex and QTc (area under the curve, 0.80 [0.76-0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (area under the curve, 0.86 [0.83-0.89]) or attainable automated patch clamping peak tail current data (area under the curve, 0.84 [0.81-0.88]). High-throughput KCNH2 variant MAVE data meaningfully contribute to variant classification at scale, whereas long QT syndrome penetrance estimates and automated patch clamping peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous KCNH2 missense variants.

Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from Vomeronasal Sensory Neurons

Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from Vomeronasal Sensory Neurons

Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from Vomeronasal Sensory Neurons

Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from Vomeronasal Sensory Neurons

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

TRPM7 contributes to pyroptosis and its involvement in status epilepticus

BackgroundPyroptosis, a novel form of programmed cell death, has been implicated in neurodegeneration diseases. However, its role in status epilepticus (SE)—a condition characterized by prolonged or repeated seizures—remains inadequately understood.MethodsSE were induced by intraperitoneal injection of pilocarpine (PILO). Neuronal excitability was assessed through electroencephalogram (EEG) recordings and patch clamp. Chromatin immunoprecipitation (ChIP) assay was applied to verify the interaction of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) protein with the promoters of Nlrp3 (the gene encoding NOD-like receptor family pyrin domain containing 3) and Trpm7 (transient receptor potential melastatin 7). To further investigate the role of TRPM7 in SE, AAV-sh-TRPM7-EGFP transfected mice and TRPM7 conditional knockout (TRPM7-CKO) mice were utilized.ResultsOur findings revealed elevated levels of IL-18 and IL-1β levels in primary epilepsy patients, along with increased expression level of the TRPM7 in SE models. Knockdown of TRPM7 alleviated neuronal damage and pyroptosis, reversing PILO-treated neuronal hyperexcitability. We demonstrated that p-STAT3 binds to the promoters of both Trpm7 and Nlrp3, modulating their transcriptions in SE. Importantly, inhibition of TRPM7 with NS8593, and inflammasome inhibition with MCC950, alleviated neuronal hyperexcitability and pyroptosis in SE. A new compound, SDUY-225, formulated based on the structure of NS8593 mitigated neuronal damage, pyroptosis, and hyperexcitability.ConclusionsTRPM7 contributes to pyroptosis in SE, establishing a positive feedback loop involving the p-STAT3/TRPM7/Zn2+/p-STAT3 signaling pathway. Findings in this study raise the possibility that targeting TRPM7 and NLRP3 represents a promising therapeutic approach for SE.

TRPM8 Mutations Associated With Persistent Pain After Surgical Injury of Corneal Trigeminal Axons.

Despite extensive efforts, the mechanisms underlying pain after axonal injury remain incompletely understood. Pain following corneal refractive surgery offers a valuable human model for investigating trigeminal axonal injury because laser-assisted in situ keratomileusis (LASIK) severs axons of trigeminal ganglion neurons innervating the cornea. While the majority of patients are pain-free shortly after surgery, a minority endure persistent postoperative ocular pain. Through genomic analysis of patients experiencing persistent postoperative ocular pain, we identified rare variants in genes encoding ion channels and receptors, including TRPM8, which codes for the menthol-sensitive and cold-sensing transient receptor potential cation channel. We conducted a profiling of 2 TRPM8 mutant variants, D665N and V915M, which were identified in patients suffering from persistent pain after LASIK surgery. We used patch-clamp and multielectrode array (MEA) recordings to investigate the biophysical and pharmacologic properties of mutant vs wild-type (WT) channels. Patch-clamp analysis shows that these mutations shift the activation curves of TRPM8 in a hyperpolarized direction, with this effect being significantly different between WT and D665N channels. In addition, both mutations significantly increase channel sensitivity to the canonical ligand, menthol. MEA recordings from transfected rat trigeminal ganglion neurons indicate that expression of D665N and V915M mutant channels increases spontaneous activity compared with WT channels. Consistent with patch-clamp results, neuronal activity in MEA recordings was increased on exposure to menthol. Collectively, our findings suggest that proexcitatory mutations of TRPM8, in the context of axonal injury within the cornea, can produce trigeminal ganglion neuron hyperexcitability that contributes to persistent postoperative ocular pain. In addition to providing additional evidence for a role of TRPM8 in human pain, our results suggest that inhibitors of this channel merit future study.

Aminophylline at clinically relevant concentrations affects inward rectifier potassium current in healthy porcine and failing human cardiomyocytes in a similar manner

Aminophylline, a bronchodilator mainly used to treat severe asthma attacks, may induce arrhythmias. Unfortunately, the underlying mechanism is not well understood. We have recently described a significant, on average inhibitory effect of aminophylline on inward rectifier potassium current IK1, known to substantially contribute to arrhythmogenesis, in rat ventricular myocytes at room temperature. This study was aimed to examine whether a similar effect may be observed under clinically relevant conditions. Experiments were performed using the whole cell patch clamp technique at 37°C on enzymatically isolated healthy porcine and failing human ventricular myocytes. The effect of clinically relevant concentrations of aminophylline (10-100µM) on IK1 did not significantly differ in healthy porcine and failing human ventricular myocytes. IK1 was reversibly inhibited by ~20 and 30% in the presence of 30 and 100µM aminophylline, respectively, at -110 mV; an analogical effect was observed at -50 mV. To separate the impact of IK1 changes on AP configuration, potentially interfering ionic currents were blocked (L-type calcium and delayed rectifier potassium currents). A significant prolongation of AP duration was observed in the presence of 100µM aminophylline in porcine cardiomyocytes which well agreed with the effect of a specific IK1 inhibitor Ba2+ (10µM) and with the result of simulations using a porcine ventricular cell model. We conclude that the observed effect of aminophylline on healthy porcine and failing human IK1 might be involved in its proarrhythmic action. To fully understand the underlying mechanism, potential aminophylline impact on other ionic currents should be explored.

Activation of hypoactive parvalbumin-positive fast-spiking interneurons restores dentate inhibition to reduce electrographic seizures in the mouse intrahippocampal kainate model of temporal lobe epilepsy

Parvalbumin-positive (PV+) GABAergic interneurons in the dentate gyrus provide powerful perisomatic inhibition of dentate granule cells (DGCs) to prevent overexcitation and maintain the stability of dentate gyrus circuits. Most dentate PV+ interneurons survive status epilepticus, but surviving PV+ interneuron mediated inhibition is compromised in the dentate gyrus shortly after status epilepticus, contributing to epileptogenesis in temporal lobe epilepsy. It is uncertain whether the impaired activity of dentate PV+ interneurons recovers at later times or if it continues for months following status epilepticus. The development of compensatory modifications related to PV+ interneuron circuits in the months following status epilepticus is unknown, although reduced dentate GABAergic inhibition persists long after status epilepticus. We employed whole-cell patch-clamp recordings from dentate PV+ interneurons and DGCs in slices from male and female sham controls and intrahippocampal kainate (IHK) treated mice that developed spontaneous seizures months after status epilepticus to study epilepsy-associated changes in dentate PV+ interneuron circuits. Electrical recordings showed that: 1) Action potential firing rates of dentate PV+ interneurons were reduced in IHK treated mice up to four months after status epilepticus; 2) spontaneous inhibitory postsynaptic currents (sIPSCs) in DGCs exhibited reduced frequency but increased amplitude in IHK treated mice; and 3) the amplitude of IPSCs in DGCs evoked by optogenetic activation of dentate PV+ cells was upregulated without changes in short-term plasticity. Video-EEG recordings revealed that IHK treated mice showed spontaneous electrographic seizures in the dentate gyrus and that chemogenetic activation of PV+ interneurons abolished electrographic seizures. Our results suggest not only that the compensatory changes in PV+ interneuron circuits develop after IHK treatment, but also that increased PV+ interneuron mediated inhibition in the dentate gyrus may compensate for cell loss and reduced intrinsic excitability of dentate PV+ interneurons to stop seizures in temporal lobe epilepsy.

The sodium/glucose cotransporter 2 inhibitor Empagliflozin inhibits long QT 3 late sodium currents in a mutation specific manner

BackgroundSodium/glucose cotransporter 2 inhibitors (SGLT2is) like empagliflozin have demonstrated cardioprotective effects in patients with or without diabetes. SGLT2is have been shown to selectively inhibit the late component of cardiac sodium current (late INa). Induction of late INa is the primary mechanism in the pathophysiology of congenital long QT syndrome type 3 (LQT3) gain-of-function mutations in the SCN5A gene encoding Nav1.5. We investigated empagliflozin's effect on late INa in thirteen known LQT3 mutations located in distinct regions of the channel. MethodsThe whole-cell patch-clamp technique was used to investigate the effect of empagliflozin on late INa in recombinantly expressed Nav1.5 channels containing different LQT3 mutations. Molecular modeling of human Nav1.5 and simulations in a mathematical model of human ventricular myocytes were used to extrapolate our experimental results to excitation-contraction coupling. ResultsEmpagliflozin selectively inhibited late INa in LQT3 mutations in the inactivation gate region of Nav1.5, without affecting peak current or channel kinetics. In contrast, empagliflozin inhibited both peak and late INa in mutations in the S4 voltage-sensing regions, altered channel gating, and slowed recovery from inactivation. Empagliflozin had no effect on late/peak INa or channel kinetics in channels with mutations in the putative empagliflozin binding region. Simulation results predict that empagliflozin may have a desirable therapeutic effect in LQT3 mutations in the inactivation gate region. ConclusionsEmpagliflozin selectively inhibits late INa, without affecting channel kinetics, in LQT3 mutations in the inactivation gate region. Empagliflozin may thus be a promising precision medicine approach for patients with specific LQT3 mutations.

Decoding taste perception: the role of electrophysiology in taste transduction mechanisms

Taste perception is a vital sensory process that helps organisms distinguish nutrients from harmful substances. It converts chemical stimuli into electrical signals through receptors on taste receptor cells within taste buds. The five primary taste modalities—sweet, salty, sour, bitter, and umami—are mediated by distinct molecular mechanisms. G-protein-coupled receptors, such as T1R and T2R families, are responsible for sweet, umami, and bitter tastes, while ion channels like epithelial sodium channels and proton-sensitive channels govern salty and sour detection. Electrophysiological techniques like patch-clamp recordings, multi-electrode arrays, and voltage-sensitive imaging have provided insights into the electrical properties of taste cells, neurotransmitter release, and neural coding models. Recent advancements in optogenetics, microfluidic devices, and nanoelectrode arrays have refined our understanding of taste transduction at the cellular and network levels. This review highlights key findings in the electrophysiology of taste, focusing on progress in understanding peripheral and central taste processing. It also discusses emerging technologies and future directions that could further advance the field and address ongoing questions about taste perception.

A deep learning approach to real-time Markov modeling of ion channel gating

The patch-clamp technique allows us to eavesdrop the gating behavior of individual ion channels with unprecedented temporal resolution. The signals arise from conformational changes of the channel protein as it makes rapid transitions between conducting and non-conducting states. However, unambiguous analysis of single-channel datasets is challenging given the inadvertently low signal-to-noise ratio as well as signal distortions caused by low-pass filtering. Ion channel kinetics are typically described using hidden Markov models (HMM), which allow conclusions on the inner workings of the protein. In this study, we present a Deep Learning approach for extracting models from single-channel recordings. Two-dimensional dwell-time histograms are computed from the idealized time series and are subsequently analyzed by two neural networks, that have been trained on simulated datasets, to determine the topology and the transition rates of the HMM. We show that this method is robust regarding noise and gating events beyond the corner frequency of the low-pass filter. In addition, we propose a method to evaluate the goodness of a predicted model by re-simulating the prediction. Finally, we tested the algorithm with data recorded on a patch-clamp setup. In principle, it meets the requirements for model extraction during an ongoing recording session in real-time.

The gating properties of Drosophila NMJ glutamate receptors and their dependence on Neto.

The Drosophila neuromuscular junction (NMJ) is a powerful genetic system that has revealed numerous conserved mechanisms for synapse development and homeostasis. The fly NMJ uses glutamate as the excitatory neurotransmitter and relies on kainate-type glutamate receptors and their auxiliary protein Neto for synapse assembly and function. However, despite decades of study, the reconstitution of NMJ glutamate receptors using heterologous systems has been achieved only recently, and there are no reports on the gating properties for the recombinant receptors. Here, using outside-out, patch clamp recordings and fast ligand application, we examine for the first time the biophysical properties of native type-A and type-B NMJ receptors in complexes with either Neto-α or Neto-β and compare them with recombinant receptors expressed in HEK293T cells. We found that type-A and type-B receptors have strikingly different gating properties that are further modulated by Neto-α and Neto-β. We captured single-channel events and revealed major differences between type-A and type-B receptors and also between Neto splice variants. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of ionotropic glutamate receptor (iGluR) desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals. KEY POINTS: We report the reconstitution of Drosophila neuromuscular junction ionotropic glutamate receptors (iGluRs) with Neto splice forms. Using outside-out patches and fast ligand application, we examine the deactivation and desensitization of the four iGluR/Neto complexes found in vivo. Expression of functional channels is absolutely dependent on Neto. Single-channel recordings revealed different lifetimes for different receptor complexes. The decay of synaptic currents is controlled by desensitization.