Fast Inactivation Research Articles

A mechanistic reinterpretation of fast inactivation in voltage-gated Na+ channel

A mechanistic reinterpretation of fast inactivation in voltage-gated Na+ channel

Functional analysis of ctenophore Shaker K+ channels: N-type inactivation in the animal roots

Here we explore the evolutionary origins of fast N-type ball-and-chain inactivation in Shaker (Kv1) K+ channels by functionally characterizing Shaker channels from the ctenophore (comb jelly) Mnemiopsis leidyi. Ctenophores are the sister lineage to other animals and Mnemiopsis has >40 Shaker-like K+ channels, but they have not been functionally characterized. We identified three Mnemiopsis channels (MlShak3–5) with N-type inactivation ball-like sequences at their N termini and functionally expressed them in Xenopus oocytes. Two of the channels, MlShak4 and MlShak5, showed rapid inactivation similar to cnidarian and bilaterian Shakers with rapid N-type inactivation, whereas MlShak3 inactivated ∼100-fold more slowly. Fast inactivation in MlShak4 and MlShak5 required the putative N-terminal inactivation ball sequences. Furthermore, the rate of fast inactivation in these channels depended on the number of inactivation balls/channel, but the rate of recovery from inactivation did not. These findings closely match the mechanism of N-type inactivation first described for Drosophila Shaker in which 1) inactivation balls on the N termini of each subunit can independently block the pore, and 2) only one inactivation ball occupies the pore binding site at a time. These findings suggest classical N-type activation evolved in Shaker channels at the very base of the animal phylogeny in a common ancestor of ctenophores, cnidarians, and bilaterians and that fast-inactivating Shakers are therefore a fundamental type of animal K+ channel. Interestingly, we find evidence from functional co-expression experiments and molecular dynamics that MlShak4 and MlShak5 do not co-assemble, suggesting that Mnemiopsis has at least two functionally independent N-type Shaker channels.

Recent advances in micro/nanomotors for antibacterial applications.

Currently, the rapid spread of multidrug-resistant bacteria derived from the indiscriminate use of traditional antibiotics poses a significant threat to public health worldwide. Moreover, established bacterial biofilms are extremely difficult to eradicate because of their high tolerance to traditional antimicrobial agents and extraordinary resistance to phagocytosis. Hence, it is of universal significance to develop novel robust and efficient antibacterial strategies to combat bacterial infections. Micro/nanomotors exhibit many intriguing properties, including enhanced mass transfer and micro-mixing resulting from their locomotion, intrinsic antimicrobial capabilities, active cargo delivery, and targeted treatment with precise micromanipulation, which facilitate the targeted delivery of antimicrobials to infected sites and their deep permeation into sites of bacterial biofilms for fast inactivation. Thus, the ideal antimicrobial activity of antibacterial micro/nanorobots makes them desirable alternatives to traditional antimicrobial treatments and has aroused extensive interest in recent years. In this review, recent advancements in antibacterial micro/nanomotors are briefly summarized, focusing on their synthetic methods, propulsion mechanism, and versatile antibacterial applications. Finally, some personal insights into the current challenges and possible future directions to translate proof-of-concept research to clinic application are proposed.

Inhibition of Cardiac Kv4.3/KChIP2 Channels by Sulfonylurea Drug Gliquidone.

The Kv4.3 channel features fast N-type inactivation and also undergoes a slow C-type inactivation. The gain-of-function mutations of Kv4.3 channels cause an inherited disease called Brugada syndrome (BrS), characterized by a shortened duration of cardiac action potential repolarization and ventricular arrhythmia. The sulfonylurea drug gliquidone, an ATP-dependent K+ channel antagonist, is widely used for the treatment of type 2 diabetes. Here, we report a novel role of gliquidone in inhibiting Kv4.3 and Kv4.3/KChIP2 channels that encode the cardiac transient outward K+ currents responsible for the initial phase of action potential repolarization. Gliquidone results in concentration-dependent inhibition of both Kv4.3 and Kv4.3/KChIP2 fast or steady-state inactivation currents with an IC50 of approximately 8 μM. Gliquidone also accelerates Kv4.3 channel inactivation and shifts the steady-state activation to a more depolarizing direction. Site-directed mutagenesis and molecular docking reveal that the residues S301 in the S4 and Y312A and L321A in the S4-S5 linker are critical for gliquidone-mediated inhibition of Kv4.3 currents, as mutating those residues to alanine significantly reduces the potency for gliquidone-mediated inhibition. Furthermore, gliquidone also inhibits a gain-of-function Kv4.3 V392I mutant identified in BrS patients in voltage- and concentration-dependent manner. Taken together, our findings demonstrate that gliquidone inhibits Kv4.3 channels by acting on the residues in the S4 and the S4-S5 linker. Therefore, gliquidone may hold repurposing potential for the therapy of Brugada syndrome. SIGNIFICANCE STATEMENT: We describe a novel role of gliquidone in inhibiting cardiac Kv4.3 currents and the channel gain-of-function mutation identified from patients with Brugada syndrome, suggesting its repurposing potential for therapy for the heart disease.

LmNaTx15, a novel scorpion toxin, enhances the activity of Nav channels and induces pain in mice

Polypeptide toxins are major bioactive components found in venomous animals. Many polypeptide toxins can specifically act on targets, such as ion channels and voltage-gated sodium (Nav) channels, in the nervous, muscle, and cardiovascular systems of the recipient to increase defense and predation efficiency. In this study, a novel polypeptide toxin, LmNaTx15, was isolated from the venom of the scorpion Lychas mucronatus, and its activity was analyzed. LmNaTx15 slowed the fast inactivation of Nav1.2, Nav1.3, Nav1.4, Nav1.5, and Nav1.7 and inhibited the peak current of Nav1.5, but it did not affect Nav1.8. In addition, LmNaTx15 altered the voltage-dependent activation and inactivation of these Nav channel subtypes. Furthermore, like site 3 neurotoxins, LmNaTx15 induced pain in mice. These results show a novel scorpion toxin with a modulatory effect on specific Nav channel subtypes and pain induction in mice. Therefore, LmNaTx15 may be a key bioactive component for scorpion defense and predation. Besides, this study provides a basis for analyzing structure-function relationships of the scorpion toxins affecting Nav channel activity.

Pharmaceuticals degradation and pathogens inactivation in municipal wastewater: A comparison among UVC photo-Fenton with chelating agents, UVC/H2O2 and ozonation

In this work, emerging advanced oxidation processes (AOPs), namely UVC photo-Fenton with two chelating agents at neutral pH, were compared for the first time to consolidated AOPs, namely, UVC/H2O2 and Ozonation (O3) in the degradation of three pharmaceuticals (PhCs) and pathogens inactivation, in real municipal wastewater (RMWW) at pilot scale. Conventional UVC based AOP (UVC/H2O2 at 50 mg/L of H2O2) was faster, being able to degrade 80% of the sum of PhCs in RMWW after 2.5 min, while the same treatment time was required to achieve 60% removal by photo-Fenton. 60% removal of the sum of PhCs in RMWW by O3 was achieved only after 75 min, at the highest dose investigated (0.45 g O3/ L h). The detection limit (1 CFU/100 mL) for pathogens inactivation was achieved after 120 min by O3 for E. coli, while more time was required for Enterococcus (10 CFU/100 mL after 120 min). Faster inactivation was observed for UVC/H2O2 (15 mg/L), the DL being achieved just within 5 and 10 min for E. coli and Enterococcus, respectively. Accordingly, UVC/H2O2 at 15 mg/L would be an effective solution for simultaneous PhCs removal and bacteria inactivation, showing better performance compared to O3 and UVC photo-Fenton like process.

Ih current stabilizes excitability in rodent DRG neurons and reverses hyperexcitability in a nociceptive neuron model of inherited neuropathic pain.

We show here that hyperpolarization-activated current (Ih ) unexpectedly acts to inhibit the activity of dorsal root ganglion (DRG) neurons expressing WT Nav1.7, the largest inward current and primary driver of DRG neuronal firing, and hyperexcitable DRG neurons expressing a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain. In this study we created a kinetic model of Ih and used it, in combination with dynamic-clamp, to study Ih function in DRG neurons. We show, for the first time, that Ih increases rheobase and reduces the firing probability in small DRG neurons, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . Our results show that Ih , due to slow gating, is not deactivated during action potentials (APs) and has a striking damping action, which reverses from depolarizing to hyperpolarizing, close to the threshold for AP generation. Moreover, we show that Ih reverses the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. In the aggregate, our results show that Ih unexpectedly has strikingly different effects in DRG neurons as compared to previously- and well-studied cardiac cells. Within DRG neurons where Nav1.7 is present, Ih reduces depolarizing sodium current inflow due to enhancement of Nav1.7 channel fast inactivation and creates additional damping action by reversal of Ih direction from depolarizing to hyperpolarizing close to the threshold for AP generation. These actions of Ih limit the firing of DRG neurons expressing WT Nav1.7 and reverse the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. KEY POINTS: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, the molecular determinants of hyperpolarization-activated current (Ih ) have been characterized as a 'pain pacemaker', and thus considered to be a potential molecular target for pain therapeutics. Dorsal root ganglion (DRG) neurons express Nav1.7, a channel that is not present in central neurons or cardiac tissue. Gain-of-function mutations (GOF) of Nav1.7 identified in inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, produce DRG neuron hyperexcitability, which in turn produces severe pain. We found that Ih increases rheobase and reduces firing probability in small DRG neurons expressing WT Nav1.7, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . We also demonstrate that Ih reverses the hyperexcitability of DRG neurons expressing a GOF Nav1.7 mutation (L858H) that causes IEM. Our results show that, in contrast to cardiac cells and CNS neurons, Ih acts to stabilize DRG neuron excitability and prevents excessive firing.

The Hodgkin-Huxley-Katz Prize Lecture: A Markov model with permeation-dependent gating that accounts for resurgent current of voltage-gated Na channels.

Many neurons that fire high-frequency action potentials express specialized voltage-gated Na channel complexes that not only conduct transient current upon depolarization, but also pass resurgent current upon repolarization. The resurgent current is associated with recovery of transient current, even at moderately negative potentials where fast inactivation is usually absorbing. The combined results of many experimental studies have led to the hypothesis that resurgent current flows upon repolarization when an endogenous blocking protein that occludes open channels at depolarized potentials is expelled by inwardly permeating Na ions. Additional observations have suggested that the position of the voltage sensor of domain IV regulates the affinity of the channel for the putative blocker. To test the effectiveness of a kinetic scheme incorporating these features, here we develop and justify a Markov model with states grounded in known Na channel conformations. Simulations were designed to investigate whether including a permeation-dependent unblocking rate constant and two open-blocked states, superimposed on conformations and voltage-sensitive movements present in all voltage-gated Na channels, is sufficient to account for the unusual gating of channels with a resurgent component. Optimizing rate constant parameters against a wide range of experimental data from cerebellar Purkinje cells demonstrates that a kinetic scheme for Na channels incorporating the novel aspects of a permeation-dependent unblock,as well as distinct high- and low-affinity blocked states, reproduces all the attributes of experimentally recorded Na currents in a physiologically plausible manner.

The fungicide tebuconazole modulates the sodium current of human NaV1.5 channels expressed in HEK293cells.

The fungicide Tebuconazole is a widely used pesticide in agriculture and may cause cardiotoxicity. In our present investigation the effect of Tebuconazole on the sodium current (INa) of human cardiac sodium channels (NaV1.5) was studied using a heterologous expression system and whole-cell patch-clamp techniques. Tebuconazole reduced the amplitude of the peak INa in a concentration- and voltage-dependent manner. At the holding potential of -120 mV the IC50 was estimated at 204.1±34.3μM, while at -80 mV the IC50 was 0.3±0.1μM. The effect of the fungicide is more pronounced at more depolarized potentials, indicating a state-dependent interaction. Tebuconazole caused a negative shift in the half-maximal inactivation voltage and delayed recovery from fast inactivation of INa. Also, it enhanced closed-state inactivation, exhibited use-dependent block in a voltage-dependent manner. Furthermore, Tebuconazole reduced the increase in late sodium current induced by the pyrethroid insecticide β-Cyfluthrin. These results suggest that Tebuconazole can interact with NaV1.5 channels and modulate INa. The observed effects may lead to decreased cardiac excitability through reduced INa availability, which could be a new mechanism of cardiotoxicity to be attributed to the fungicide.

Molecular action mechanisms of two novel and selective calcium release-activated calcium channel antagonists

The prototypical calcium release-activated calcium (CRAC) channel, composed of STIM1 and Orai1, is a sought-after drug target for treating autoimmune disorders. Herein, we identified two novel and selective CRAC channel inhibitors, the indole-like compound C63368 and pyrazole core-containing compound C79413, potently and reversibly inhibiting the CRAC channel with low micromolar IC50s and sparing various off-target ion channels. These two compounds did not inhibit STIM1 activation or its coupling with Orai1, nor did they affect the channel's calcium-dependent fast inactivation. Instead, they directly acted on the Orai1 protein, with the channel's pore geometry profoundly affecting their potencies. In vitro, C63368 and C79413 effectively inhibited Jurkat cell proliferation and cytokines production in human T lymphocytes. Intragastric administration of C63368 and C79413 to mice yielded great therapeutic benefits in psoriasis and colitis animal models of autoimmune disorders, reducing serum cytokines production and significantly relieving pathological symptoms. It's worth noting, that this study provided the first insight into the characterization and mechanistic investigation of an indole-like CRAC channel antagonist. Altogether, the identification of these two highly selective CRAC channel antagonists, coupled with the elucidation of their action mechanisms, not only provides valuable template molecules but also offers profound insights for drug development targeting the CRAC channel.

Selenomethionine mis-incorporation and redox-dependent voltage-gated sodium channel gain of function.

Selenomethionine (SeMet) readily replaces methionine (Met) residues in proteins during translation. Long-term dietary SeMet intake results in the accumulation of the amino acid in tissue proteins. Despite the high rates of SeMet incorporation in proteins and its stronger susceptibility to oxidation compared to Met, little is known about the effect of SeMet mis-incorporation on electrical excitability and ion channels. Fast inactivation of voltage-gated sodium (NaV ) channels is essential for exact action potential shaping with even minute impairment of inactivation resulting in a plethora of adverse phenotypes. Met oxidation of the NaV channel inactivation motif (Ile-Phe-Met) and further Met residues causes a marked loss of inactivation. Here, we examined the impact of SeMet mis-incorporation on the function of NaV channels. While extensive SeMet incorporation into recombinant rat NaV 1.4 channels preserved their normal function, it greatly sensitized the channels to mild oxidative stress, resulting in loss of inactivation and diminished maximal current, both reversible by dithiothreitol-induced reduction. SeMet incorporation similarly affected human NaV 1.4, NaV 1.2, NaV 1.5, and NaV 1.7. In mouse dorsal root ganglia (DRG) neurons, 1 day of SeMet exposure exacerbated the oxidation-mediated broadening of action potentials. SeMet-treated DRGs also exhibited a stronger increase in the persistent NaV current in response to oxidation. SeMet incorporation in NaV proteins coinciding with oxidative insults may therefore result in hyperexcitability pathologies, such as cardiac arrhythmias and neuropathies, like congenital NaV channel gain-of-function mutations.

A mechanistic reinterpretation of fast inactivation in voltage-gated Na+ channels

The hinged-lid model was long accepted as the canonical model for fast inactivation in Nav channels. It predicts that the hydrophobic IFM motif acts intracellularly as the gating particle that binds and occludes the pore during fast inactivation. However, the observation in recent high-resolution structures that the bound IFM motif is located far from the pore, contradicts this preconception. Here, we provide a mechanistic reinterpretation of fast inactivation based on structural analysis and ionic/gating current measurements. We demonstrate that in Nav1.4 the final inactivation gate is comprised of two hydrophobic rings at the bottom of S6 helices. These rings function in series and close downstream of IFM binding. Reducing the volume of the sidechain in both rings leads to a partially conductive, leaky inactivated state and decreases the selectivity for Na+ ion. Altogether, we present an alternative molecular framework to describe fast inactivation.

Cold and warmth intensify pain-linked sodium channel gating effects and persistent currents.

Voltage-gated sodium channels (Nav) are key players in excitable tissues with the capability to generate and propagate action potentials. Mutations in the genes encoding Navs can lead to severe inherited diseases, and some of these so-called channelopathies show temperature-sensitive phenotypes, for example, paramyotonia congenita, Brugada syndrome, febrile seizure syndromes, and inherited pain syndromes like erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). Nevertheless, most investigations of mutation-induced gating effects have been conducted at room temperature, and thus the role of cooling or warming in channelopathies remains poorly understood. Here, we investigated the temperature sensitivity of four Nav subtypes: Nav1.3, Nav1.5, Nav1.6, and Nav1.7, and two mutations in Nav1.7 causing IEM (Nav1.7/L823R) and PEPD (Nav1.7/I1461T) expressed in cells of the human embryonic kidney cell line using an automated patch clamp system. Our experiments at 15°C, 25°C, and 35°C revealed a shift of the voltage dependence of activation to more hyperpolarized potentials with increasing temperature for all investigated subtypes. Nav1.3 exhibited strongly slowed inactivation kinetics compared with the other subtypes that resulted in enhanced persistent current, especially at 15°C, indicating a possible role in cold-induced hyperexcitability. Impaired fast inactivation of Nav1.7/I1461T was significantly enhanced by a cooling temperature of 15°C. The subtype-specific modulation as well as the intensified mutation-induced gating changes stress the importance to consider temperature as a regulator for channel gating and its impact on cellular excitability as well as disease phenotypes.

Anticonvulsant effects of cenobamate in chemically and electrically induced seizure models in rodents

BackgroundCenobamate is an antiseizure medication used to treat partial-onset (focal) seizures. It is a molecule with one chiral center and a unique dual mechanism of action: enhancement of fast and slow inactivation of sodium channels with preferential inhibition of the persistent current and positive allosteric modulation of GABAA receptor-mediated ion channels. Aims/MethodsAnticonvulsant effects of cenobamate (YKP3089; R-enantiomer), YKP3090 (S-enantiomer), and YKP1983 (racemate) were evaluated in chemically and electrically induced focal and generalized seizure models in rodents. The Genetic Absence Epilepsy Rat from Strasbourg (GAERS) model examined the effect of cenobamate on spike-wave seizures. Motor coordination was assessed with rotarod tests and minimal motor impairment exams. ResultsEarly in development, cenobamate was found to have activity in focal and generalized seizure models in animals and was selected for continued development. Cenobamate prevented seizures in a dose-dependent manner, prevented seizure spread, and increased seizure threshold without potentiating seizure initiation or the development of tolerance to its anticonvulsant effects. In contrast, YKP3090 and YKP1983 were only effective against generalized tonic-clonic seizures. Cenobamate also protected mice from 6 Hz psychomotor-induced seizures. Cenobamate showed significant dose-dependent reductions in the number and cumulative duration of spike-and-wave discharges in the GAERS model. DiscussionCenobamate showed efficacy or efficacy signals in all animal models of epilepsy tested with a favorable risk-versus-benefit ratio, supporting its clinical use in the treatment of partial-onset (focal) seizures in adults and warranting further clinical research in generalized seizures and absence seizures.

The opioid tramadol blocks the cardiac sodium channel Nav1.5 in HEK293 cells.

Opioids are associated with increased risk of sudden cardiac death. This may be due to their effects on the cardiac sodium channel (Nav1.5) current. In the present study, we aim to establish whether tramadol, fentanyl, or codeine affects Nav1.5 current. Using whole-cell patch-clamp methodology, we studied the effects of tramadol, fentanyl, and codeine on currents of human Nav1.5 channels stably expressed in HEK293 cells and on action potential (AP) properties of freshly isolated rabbit ventricular cardiomyocytes. In fully available Nav1.5 channels (holding potential -120 mV), tramadol exhibited inhibitory effects on Nav1.5 current in a concentration-dependent manner with an IC50 of 378.5 ± 33.2 µm. In addition, tramadol caused a hyperpolarizing shift of voltage-gated (in)activation and a delay in recovery from inactivation. These blocking effects occurred at lower concentrations in partially inactivated Nav1.5 channels: during partial fast inactivation (close-to-physiological holding potential -90 mV), IC50 of Nav1.5 block was 4.5 ± 1.1 μm, while it was 16 ± 4.8 μm during partial slow inactivation. The tramadol-induced changes on Nav1.5 properties were reflected by a reduction in AP upstroke velocity in a frequency-dependent manner. Fentanyl and codeine had no effect on Nav1.5 current, even when tested at lethal concentrations. Tramadol reduces Nav1.5 currents, in particular, at close-to-physiological membrane potentials. Fentanyl and codeine have no effects on Nav1.5 current.

Dysfunctional sodium channel kinetics as a novel epilepsy mechanism in chromosome 15q11-q13 duplication syndrome.

Duplication of the maternal chromosome 15q11.2-q13.1 region causes Dup15q syndrome, a highly penetrant neurodevelopmental disorder characterized by severe autism and refractory seizures. Although UBE3A, the gene encoding the ubiquitin ligase E3A, is thought to be the main driver of disease phenotypes, the cellular and molecular mechanisms that contribute to the development of the syndrome are yet to be determined. We previously established the necessity of UBE3A overexpression for the development of cellular phenotypes in human Dup15q neurons, including increased action potential firing and increased inward current density, which prompted us to further investigate sodium channel kinetics. We used a Dup15q patient-derived induced pluripotent stem cell line that was CRISPR-edited to remove the supernumerary chromosome and create an isogenic control line. We performed whole cell patch clamp electrophysiology on Dup15q and corrected control neurons at two time points of in vitro development. Compared to corrected neurons, Dup15q neurons showed increased sodium current density and a depolarizing shift in steady-state inactivation. Moreover, onset of slow inactivation was delayed, and a faster recovery from both fast and slow inactivation processes was observed in Dup15q neurons. A fraction of sodium current in Dup15q neurons (~15%) appeared to be resistant to slow inactivation. Not unexpectedly, a higher fraction of persistent sodium current was also observed in Dup15q neurons. These phenotypes were modulated by the anticonvulsant drug rufinamide. Sodium channels play a crucial role in the generation of action potentials, and sodium channelopathies have been uncovered in multiple forms of epilepsy. For the first time, our work identifies in Dup15q neurons dysfunctional inactivation kinetics, which have been previously linked to multiple forms of epilepsy. Our work can also guide therapeutic approaches to epileptic seizures in Dup15q patients and emphasize the role of drugs that modulate inactivation kinetics, such as rufinamide.

Cannabidiol inhibits Nav channels through two distinct binding sites

Cannabidiol (CBD), a major non-psychoactive phytocannabinoid in cannabis, is an effective treatment for some forms of epilepsy and pain. At high concentrations, CBD interacts with a huge variety of proteins, but which targets are most relevant for clinical actions is still unclear. Here we show that CBD interacts with Nav1.7 channels at sub-micromolar concentrations in a state-dependent manner. Electrophysiological experiments show that CBD binds to the inactivated state of Nav1.7 channels with a dissociation constant of about 50 nM. The cryo-EM structure of CBD bound to Nav1.7 channels reveals two distinct binding sites. One is in the IV-I fenestration near the upper pore. The other binding site is directly next to the inactivated “wedged” position of the Ile/Phe/Met (IFM) motif on the short linker between repeats III and IV, which mediates fast inactivation. Consistent with producing a direct stabilization of the inactivated state, mutating residues in this binding site greatly reduced state-dependent binding of CBD. The identification of this binding site may enable design of compounds with improved properties compared to CBD itself.

The volatile anesthetic isoflurane differentially inhibits voltage-gated sodium channel currents between pyramidal and parvalbumin neurons in the prefrontal cortex.

How volatile anesthetics work remains poorly understood. Modulations of synaptic neurotransmission are the direct cellular mechanisms of volatile anesthetics in the central nervous system. Volatile anesthetics such as isoflurane may reduce neuronal interaction by differentially inhibiting neurotransmission between GABAergic and glutamatergic synapses. Presynaptic voltage-dependent sodium channels (Nav), which are strictly coupled with synaptic vesicle exocytosis, are inhibited by volatile anesthetics and may contribute to the selectivity of isoflurane between GABAergic and glutamatergic synapses. However, it is still unknown how isoflurane at clinical concentrations differentially modulates Nav currents between excitatory and inhibitory neurons at the tissue level. In this study, an electrophysiological recording was applied in cortex slices to investigate the effects of isoflurane on Nav between parvalbumin (PV+) and pyramidal neurons in PV-cre-tdTomato and/or vglut2-cre-tdTomato mice. Isoflurane at clinically relevant concentrations produced a hyperpolarizing shift in the voltage-dependent inactivation and slowed the recovery time from the fast inactivation in both cellular subtypes. Since the voltage of half-maximal inactivation was significantly depolarized in PV+ neurons compared to that of pyramidal neurons, isoflurane inhibited the peak Nav currents in pyramidal neurons more potently than those of PV+ neurons (35.95 ± 13.32% vs. 19.24 ± 16.04%, P = 0.036 by the Mann-Whitney test). Isoflurane differentially inhibits Nav currents between pyramidal and PV+ neurons in the prefrontal cortex, which may contribute to the preferential suppression of glutamate release over GABA release, resulting in the net depression of excitatory-inhibitory circuits in the prefrontal cortex.

Biallelic structural variations within FGF12 detected by long-read sequencing in epilepsy.

We discovered biallelic intragenic structural variations (SVs) in FGF12 by applying long-read whole genome sequencing to an exome-negative patient with developmental and epileptic encephalopathy (DEE). We also found another DEE patient carrying a biallelic (homozygous) single-nucleotide variant (SNV) in FGF12 that was detected by exome sequencing. FGF12 heterozygous recurrent missense variants with gain-of-function or heterozygous entire duplication of FGF12 are known causes of epilepsy, but biallelic SNVs/SVs have never been described. FGF12 encodes intracellular proteins interacting with the C-terminal domain of the alpha subunit of voltage-gated sodium channels 1.2, 1.5, and 1.6, promoting excitability by delaying fast inactivation of the channels. To validate the molecular pathomechanisms of these biallelic FGF12 SVs/SNV, highly sensitive gene expression analyses using lymphoblastoid cells from the patient with biallelic SVs, structural considerations, and Drosophila in vivo functional analysis of the SNV were performed, confirming loss-of-function. Our study highlights the importance of small SVs in Mendelian disorders, which may be overlooked by exome sequencing but can be detected efficiently by long-read whole genome sequencing, providing new insights into the pathomechanisms of human diseases.

Growth and Non-Thermal Inactivation of Staphylococcus aureus in Sliced Dry-Cured Ham in Relation to Water Activity, Packaging Type and Storage Temperature.

Dry-cured ham (DCH) could support the growth of Staphylococcus aureus as a halotolerant bacterium, which may compromise the shelf-stability of the product according to the growth/no growth boundary models and the physicochemical parameters of commercial DCH. In the present study, the behavior of S. aureus is evaluated in sliced DCH with different water activity (aw 0.861-0.925), packaged under air, vacuum, or modified atmosphere (MAP), and stored at different temperatures (2-25 °C) for up to 1 year. The Logistic and the Weibull models were fitted to data to estimate the primary kinetic parameters for the pathogen Log10 increase and Log10 reduction, respectively. Then, polynomial models were developed as secondary models following their integration into the primary Weibull model to obtain a global model for each packaging. Growth was observed for samples with the highest aw stored at 20 and 25 °C in air-packaged DCH. For lower aw, progressive inactivation of S. aureus was observed, being faster at the lowest temperature (15 °C) for air-packaged DCH. In contrast, for vacuum and MAP-packaged DCH, a higher storage temperature resulted in faster inactivation without a significant effect of the product aw. The results of this study clearly indicate that the behavior of S. aureus is highly dependent on factors such as storage temperature, packaging conditions and product aw. The developed models provide a management tool for evaluating the risk associated with DCH and for preventing the development of S. aureus by selecting the most appropriate packaging according to aw range and storage temperature.