Channel Activity Research Articles

Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins.

Store-operated Ca2+ entry is a mechanism controlled by the filling state of the intracellular Ca2+ stores, predominantly the endoplasmic reticulum (ER), where ER-resident proteins STIM1 and STIM2 orchestrate the activation of Orai channels in the plasma membrane, and Orai1 playing a predominant role. Two forms of Orai1, Orai1α and Orai1β, have been identified, which arises the question whether they are equally regulated by STIM proteins. We demonstrate that STIM1 preferentially activates Orai1α over STIM2, yet both STIM proteins similarly activate Orai1β. Under resting conditions, there is a pronounced association between STIM2 and Orai1α. STIM1 and STIM2 are also shown to influence the protein levels of the Orai1 variants, independently of Ca2+ influx, via lysosomal degradation. Interestingly, Orai1α and Orai1β appear to selectively regulate the protein level of STIM1, but not STIM2. These observations offer crucial insights into the regulatory dynamics between STIM proteins and Orai1 variants, enhancing our understanding of the intricate processes that fine-tune intracellular Ca2+ signaling.

Ligand-Coupled Conformational Changes in a Cyclic Nucleotide-Gated Ion Channel Revealed by Time-Resolved Transition Metal Ion FRET.

Allosteric regulation is pivotal for the function of most proteins, especially ion channels like the cyclic nucleotide-binding domain (CNBD) channels. This study examines the allosteric mechanism of ligand binding in the C-terminal region of the prokaryotic CNBD ion channel SthK using steady-state and time-resolved tmFRET. We uncovered significant structural and energetic changes induced by ligand binding with the full-agonist cAMP and the weak partial agonist cGMP. Our approach also highlights the effectiveness of using fluorescence lifetimes to reveal conformational heterogeneity and free energy changes in proteins. These findings deepen our understanding of CNBD channel activation overall and lay the groundwork for a more comprehensive characterization of the effects of mutations and pharmacological agents in these channels.

Nicorandil antiallodynic activity in a model of neuropathic pain is associated with the activation of ATP-dependent potassium channels and opioidergic pathways, and reduced production of cytokines and neutrophils recruitment in paw, sciatic nerve, and dorsal root ganglia.

Recently, we demonstrated that nicorandil inhibits mechanical allodynia induced by paclitaxel. In the present study, we evaluated the effect induced by nicorandil in a model of neuropathic pain induced by chronic constriction injury (CCI) in mice. We also investigated putative mechanisms underlying such an effect. CCI was induced by three ligatures of the left sciatic nerve. Mechanical allodynia was evaluated by measuring the paw withdrawal threshold with an electronic von Frey apparatus. Concentrations of cytokines and myeloperoxidase activity were determined in the paw tissue, sciatic nerve, and dorsal root ganglia (DRG). Oral administration of two doses of nicorandil (150mg/kg po), but not equimolar doses of nicotinamide or nicotinic acid, attenuated mechanical allodynia induced by CCI. Nicorandil activity was reduced by previous administration of glibenclamide (40mg/kg) or naltrexone (5mg/kg or 10mg/kg). Two doses of nicorandil (150mg/kg, po) reduced tumor necrosis factor-α, interleukin-1β and interleukin-6, but not CXCL-1, concentrations in the paw tissue of CCI mice. Two doses of nicorandil (150mg/kg, po) reduced concentrations of all these mediators in the sciatic nerve and DRG. Two doses of nicorandil (150mg/kg, po) also reduced the myeloperoxidase activity in the paw tissue, sciatic nerve, and DRG. Nicorandil exhibits antiallodynic activity in a model of neuropathic pain induced by CCI. Inhibition of cytokines production and reduction of neutrophils recruitment in paw tissue, sciatic nerve, and DRG as well as activation of ATP-dependent potassium channels and opioidergic pathways, underlie nicorandil antiallodynic activity.

Activation mechanism and novel binding sites of the BKCa channel activator CTIBD.

The large-conductance calcium-activated potassium (BKCa) channel, which is crucial for urinary bladder smooth muscle relaxation, is a potential target for overactive bladder treatment. Our prior work unveiled CTIBD as a promising BKCa channel activator, altering V 1/2 and G max This study investigates CTIBD's activation mechanism, revealing its independence from the Ca2+ and membrane voltage sensing of the BKCa channel. Cryo-electron microscopy disclosed that two CTIBD molecules bind to hydrophobic regions on the extracellular side of the lipid bilayer. Key residues (W22, W203, and F266) are important for CTIBD binding, and their replacement with alanine reduces CTIBD-mediated channel activation. The triple-mutant (W22A/W203A/F266A) channel showed the smallest V 1/2 shift with a minimal impact on activation and deactivation kinetics by CTIBD. At the single-channel level, CTIBD treatment was much less effective at increasing P o in the triple mutant, mainly because of a drastically increased dissociation rate compared with the WT. These findings highlight CTIBD's mechanism, offering crucial insights for developing small-molecule treatments for BKCa-related pathophysiological conditions.

Impairment of microvascular endothelial Kir2.1 channels contributes to endothelial dysfunction in human hypertension.

Hypertension is associated with decreased endothelial function through reduced contributions of nitric oxide (NO). We previously discovered that flow-induced NO production in resistance arteries of mice and humans critically depends on endothelial inwardly rectifying K+ (Kir2.1) channels. The goal of this study was to establish whether these channels contribute to the impairment of endothelial function, measured by flow-induced vasodilation (FIV) in peripheral resistance arteries of humans with hypertension. We measured FIV in vessels isolated from subcutaneous fat biopsies from 32 subjects: normotensive [n = 19; 30.6 ± 9.8 yr old; systolic blood pressure (SBP): 115.2 ± 7 mmHg; diastolic blood pressure (DBP): 75.3 ± 5.7 mmHg] and hypertensive (n = 13; 45.3 ± 15.3 yr old; SBP: 146.1 ± 15.2 mmHg; DBP: 94.4 ± 6.9 mmHg). Consistent with previous studies, we find that FIV is impaired in hypertensive adults as demonstrated by a significant reduction in FIV when compared with the normotensive adults. Furthermore, our data suggest that the impairment of FIV in hypertensive adults is partially attributed to a reduction in Kir2.1-dependent vasodilation. Specifically, we show that blocking Kir2.1 with ML133 or functionally downregulating Kir2.1 with endothelial-specific adenoviral vector containing dominant-negative Kir2.1 (dnKir2.1) result in a significant reduction in FIV in normotensive subjects but with a smaller effect in hypertensive adults. The Kir2.1-dependent vasodilation was negatively correlated to both SBP and DBP, indicating that the Kir2.1 contribution to FIV decreases as blood pressure increases. In addition, we show that exposing vessels from normotensive adults to acute high-pressure results in loss of Kir2.1 contribution, as high pressure impairs vasodilation. No effect is seen when these vessels were incubated with dnKir2.1. Overexpressing wtKir2.1 in the endothelium resulted in some improvement in vasodilation in arteries from all participants, with a greater recovery in hypertensive adults. Our data suggest that hypertension-induced suppression of Kir2.1 is an important mechanism underlying endothelial dysfunction in hypertension.NEW & NOTEWORTHY Impairment of endothelial function under high blood pressure is linked to the loss of inwardly rectifying K+ (Kir2.1) channels activity in human resistance arteries, leading to a reduction in flow-induced vasodilation and possibly leading to a vicious cycle between elevation of blood pressure, and further impairment of Kir2.1 function and flow-induced vasodilation.

Unraveling the dynamics of firing patterns for neurons with impairment of sodium channels.

Various factors such as mechanical trauma, chemical trauma, local ischemia, and inflammation can impair voltage-gated sodium channels (Nav) in neurons. These impairments lead to a distinctive leftward shift in the activation and inactivation curves of voltage-gated sodium channels. The resulting sodium channel impairments in neurons are known to affect firing patterns, which play a significant role in neuronal activities within the nervous system. However, the underlying dynamic mechanism for the emergence of these firing patterns remains unclear. In this study, we systematically investigated the effects of sodium channel dysfunction on individual neuronal dynamics and firing patterns. By employing codimension-1 bifurcation analysis, we revealed the underlying dynamical mechanism responsible for the generation of different firing patterns. Additionally, through codimension-2 bifurcation analysis, we theoretically determined the distribution of firing patterns on different parameter planes. Our results indicate that the firing patterns of impaired neurons are regulated by multiple parameters, with firing pattern transitions caused by the degree of sodium channel impairment being more diverse than those caused by the ratio of impaired sodium channel and current. Furthermore, we observed that the firing pattern of tonic firing is more likely to be the norm in impaired sodium channel neurons, providing valuable insights into the signaling of impaired neurons. Overall, our findings highlight the intricate relationships among sodium channel impairments, neuronal dynamics, and firing patterns, shedding light on the impact of disruptions in ion concentration gradients on neuronal function.

Melatonin inhibits voltage-gated potassium KV4.2 channels and negatively regulates melatonin secretion in rat pineal glands.

Melatonin is synthesized in and secreted from the pineal glands and regulates circadian rhythms. Although melatonin has been reported to modulate the activity of ion channels in several tissues, its effects on pineal ion channels remain unclear. In the present study, the effects of melatonin on voltage-gated K+ (KV) channels, which play a role in regulating the resting membrane potential, were examined in rat pinealocytes. The application of melatonin reduced pineal KV currents in a concentration-dependent manner (IC50 = 309 µM). An expression analysis revealed that KV4.2 channels were highly expressed in rat pineal glands. Melatonin-sensitive currents were abolished by the small interfering RNA knockdown of KV4.2 channels in rat pinealocytes. In human embryonic kidney 293 (HEK293) cells expressing KV4.2 channels, melatonin decreased outward currents (IC50 = 479 µM). Inhibitory effects were mediated by a shift in the voltage dependence of steady-state inactivation in a hyperpolarizing direction. This inhibition was observed even in the presence of 100 nM luzindole, an antagonist of melatonin receptors. Melatonin also blocked the activity of KV4.3, KV1.1, and KV1.5 channels in reconstituted HEK293 cells. The application of 1 mM melatonin caused membrane depolarization in rat pinealocytes. Furthermore, KV4.2 channel inhibition by 5 mM 4-aminopyridine attenuated melatonin secretion induced by 1 µM noradrenaline in rat pineal glands. These results strongly suggest that melatonin directly inhibited KV4.2 channels and caused membrane depolarization in pinealocytes, resulting in a decrease in melatonin secretion through parasympathetic signaling pathway. This mechanism may function as a negative-feedback mechanism of melatonin secretion in pineal glands. NEW & NOTEWORTHY Melatonin is a hormone that is synthesized in and secreted from the pineal glands, which regulates circadian rhythms. However, the effects of melatonin on pineal ion channels remain unclear. The present study demonstrated that melatonin directly inhibited voltage-gated potassium KV4.2 channels, which are highly expressed in rat pinealocytes, and induced membrane depolarization, resulting in a decrease in melatonin secretion. This mechanism may function as a negative-feedback mechanism of melatonin secretion in pineal glands.

Prostaglandin E2 Receptor 4 Contributes to Regulating Epithelial Sodium Channel (ENaC) Activity in the Kidney Collecting Duct

Prostaglandin E2 Receptor 4 Contributes to Regulating Epithelial Sodium Channel (ENaC) Activity in the Kidney Collecting Duct

TRESK channel activation ameliorates migraine-like pain via modulation of CGRP release from the trigeminovascular system and meningeal mast cells in experimental migraine models

AimsAccumulating evidence indicates the involvement of TRESK potassium channels in migraine, however, effects of TRESK activation on migraine-related mechanisms remain unclear. We explored effects of TRESK channel modulation on migraine-related behavioral and molecular markers in in-vivo and ex-vivo rat models of migraine. Main methodsThe selective TRESK activator cloxyquin at different doses, the TRESK inhibitor A2764, and the migraine drug sumatriptan were tested alone or in different combinations in nitroglycerin (NTG)-induced in-vivo model, and in ex-vivo meningeal, trigeminal ganglion and brainstem preparations in which CGRP release was induced by capsaicin. Mechanical allodynia, CGRP and c-fos levels in trigeminovascular structures and meningeal mast cells were evaluated. Key findingsCloxyquin attenuated NTG-induced mechanical allodynia, brainstem c-fos and CGRP levels, trigeminal ganglion CGRP levels and meningeal mast cell degranulation and number, in-vivo. It also diminished capsaicin-induced CGRP release from ex-vivo meningeal, trigeminal ganglion and brainstem preparations. Specific TRESK inhibitor A2764 abolished all effects of cloxyquin in in-vivo and ex-vivo. Combining cloxyquin and sumatriptan exerted a synergistic effect ex-vivo, but not in-vivo. SignificanceOur findings provide the experimental evidence for the anti-migraine effect of TRESK activation in migraine-like conditions. The modulation of TRESK channels may therefore be an attractive alternative strategy to relieve migraine pain.

Characterization of a novel variant in KCNJ16, encoding Kir5.1 channel.

The essential role of the inwardly rectifying potassium channel Kir5.1 (KCNJ16) in controlling electrolyte homeostasis and blood pressure has been demonstrated in human and animal studies. Previous studies have identified several bi-allelic mutations of KCNJ16 in humans, causing severe hypokalemia, renal salt wasting, and disturbed acid-base homeostasis. Here, we identified a novel homozygous variant of KCNJ16, I26T, in an Amish patient affected with polydipsia, developmental delay, and chronic metabolic acidosis with low serum bicarbonate concentration. Subsequently, we generated the rat model with I26T mutation using Dahl salt-sensitive rat (I26T rat) to characterize this variant. The male mutant rats displayed similar blood pressure and electrolyte homeostasis under baseline and with a high salt (4% NaCl) challenge. Blood pH, HCO3 - and renal damage also remained similar between WT and I26T rats after high salt challenge. Additionally, single-channel patch clamp analysis revealed similar channel activity in CHO cells overexpressed with WT and I26T mutant Kir4.1/5.1 channels. In summary, this study reported a novel variant in KCNJ16, namely I26T, which is likely a benign variant and not associated with pathologic phenotype in either human or Dahl salt-sensitive rats, indicating that the type/location of variant should be considered when diagnosing and treating patients with KCNJ16 mutations.

Voltage- and Ca2+-inducible PLC activity for analyzing PI(4,5)P2 sensitivity of ion channels in Xenopus oocytes.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key membrane lipid regulating various ion channel activities. Currently, several molecular tools are used to modulate PIP2 levels, each of which has distinct advantages and drawbacks. In this study, we proposed a novel methodology using heterologous Xenopus oocytes to precisely manipulate PIP2 levels using phospholipase C (PLC)-ζ, which hydrolyzes PIP2. Xenopus oocytes injected with PLCζ exhibited notable hyperpolarization-induced Ca2+ influx driven by the increased driving force of Ca2+. High Ca2+ sensitivity of PLCζ facilitated hyperpolarization-induced PLC activity in Xenopus oocytes that was voltage- and Ca2+-dependent. This study demonstrated the regulatory capacity of PLCζ in modulating PIP2-sensitive ion channels, such as the KCNQ2/3 and GIRK channels, in a voltage- and Ca2+-dependent manner. Moreover, activation pathway of PLCζ only requires a two-electrode voltage clamp setup, making it a convenient molecular tool to manipulate PIP2 levels in combination with a voltage-sensing phosphatase (VSP). PLCζ has distinct characteristics and advantages compared to VSP: (1) Hyperpolarization, but not depolarization, reduced the PIP2 levels, (2) PIP2 levels were decreased without any increase in phosphatidylinositol 4-monophosphate (PIP) levels, and (3) PIP2 levels were reduced by Ca2+ administration. Therefore, PLCζ effectively supports understanding how PIP2 regulates ion channels, alongside VSP. Overall, this study highlights the unique characteristics of PLCζ and its distinct advantages in analyzing ion channel regulation by PIP2 and the PLC pathway in Xenopus oocytes.

Going with the flow: New insights regarding flow induced K+ secretion in the distal nephron.

K+ secretion in the distal nephron has a critical role in K+ homeostasis and is the primary route by which K+ is lost from the body. Renal K+ secretion is enhanced by increases in dietary K+ intake and by increases in tubular flow rate in the distal nephron. This review addresses new and important insights regarding the mechanisms underlying flow-induced K+ secretion (FIKS). While basal K+ secretion in the distal nephron is mediated by renal outer medullary K+ (ROMK) channels in principal cells (PCs), FIKS is mediated by large conductance, Ca2+/stretch activated K+ (BK) channels in intercalated cells (ICs), a distinct cell type. BK channel activation requires an increase in intracellular Ca2+ concentration ([Ca2+]i), and both PCs and ICs exhibit increases in [Ca2+]i in response to increases in tubular fluid flow rate, associated with an increase in tubular diameter. PIEZO1, a mechanosensitive, nonselective cation channel, is expressed in the basolateral membranes of PCs and ICs, where it functions as a mechanosensor. The loss of flow-induced [Ca2+]i transients in ICs and BK channel-mediated FIKS in microperfused collecting ducts isolated from mice with IC-specific deletion of Piezo1 in the CCD underscores the importance of PIEZO1 in the renal regulation of K+ transport.

Discovery of E0199: A novel compound targeting both peripheral NaV and KV7 channels to alleviate neuropathic pain

This research study focuses on addressing the limitations of current neuropathic pain (NP) treatments by developing a novel dual-target modulator, E0199, targeting both NaV1.7, NaV1.8, and NaV1.9 and KV7 channels, a crucial regulator in controlling NP symptoms. The objective of the study was to synthesize a compound capable of modulating these channels to alleviate NP. Through an experimental design involving both in vitro and in vivo methods, E0199 was tested for its efficacy on ion channels and its therapeutic potential in a chronic constriction injury (CCI) mouse model. The results demonstrated that E0199 significantly inhibited NaV1.7, NaV1.8, and NaV1.9 channels with a particularly low half maximal inhibitory concentration (IC50) for NaV1.9 by promoting sodium channel inactivation, and also effectively increased KV7.2/7.3, KV7.2, and KV7.5 channels, excluding KV7.1 by promoting potassium channel activation. This dual action significantly reduced the excitability of dorsal root ganglion neurons and alleviated pain hypersensitivity in mice at low doses, indicating a potent analgesic effect without affecting heart and skeletal muscle ion channels critically. The safety of E0199 was supported by neurobehavioral evaluations. Conclusively, E0199 represents a ground-breaking approach in NP treatment, showcasing the potential of dual-target small-molecule compounds in providing a more effective and safe therapeutic option for NP. This study introduces a promising direction for the future development of NP therapeutics.

Amyloid-β oligomers trigger sex-dependent inhibition of GIRK channel activity in hippocampal neurons in mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by amyloid plaques and cognitive decline, the latter of which is thought to be driven by soluble oligomeric amyloid-β (oAβ). The dysregulation of G protein-gated inwardly rectifying K+ (GIRK; also known as Kir3) channels has been implicated in rodent models of AD. Here, seeking mechanistic insights, we uncovered a sex-dependent facet of GIRK-dependent signaling in AD-related amyloid pathophysiology. Synthetic oAβ1-42 suppressed GIRK-dependent signaling in hippocampal neurons from male mice, but not from female mice. This effect required cellular prion protein, the receptor mGluR5, and production of arachidonic acid by the phospholipase PLA2. Although oAβ suppressed GIRK channel activity only in male hippocampal neurons, intrahippocampal infusion of oAβ or genetic suppression of GIRK channel activity in hippocampal pyramidal neurons impaired performance on a memory test in both male and female mice. Moreover, genetic enhancement of GIRK channel activity in hippocampal pyramidal neurons blocked oAβ-induced cognitive impairment in both male and female mice. In APP/PS1 AD model mice, GIRK-dependent signaling was diminished in hippocampal CA1 pyramidal neurons from only male mice before cognitive deficit was detected. However, enhancing GIRK channel activity rescued cognitive deficits in older APP/PS1 mice of both sexes. Thus, whereas diminished GIRK channel activity contributes to cognitive deficits in male mice with increased oAβ burden, enhancing its activity may have therapeutic potential for both sexes.

Direct conversion of human umbilical cord mesenchymal stem cells into dopaminergic neurons for Parkinson's disease treatment

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor deficits due to the depletion of nigrostriatal dopamine. Stem cell differentiation therapy emerges as a promising treatment option for sustained symptom relief. In this study, we successfully developed a one-step differentiation system using the YFBP cocktail (Y27632, Forskolin, SB431542, and SP600125) to effectively convert human umbilical cord mesenchymal stem cells (hUCMSCs) into dopaminergic neurons without genetic modification. This approach addresses the challenge of rapidly and safely generating functional neurons on a large scale. After a 7-day induction period, over 80 % of the cells were double-positive for TUBB3 and NEUN. Transcriptome analysis revealed the dual roles of the cocktail in inducing fate erasure in mesenchymal stem cells and activating the neuronal program. Notably, these chemically induced cells (CiNs) did not express HLA class II genes, preserving their immune-privileged status. Further study indicated that YFBP significantly downregulated p53 signaling and accelerated the differentiation process when Pifithrin-α, a p53 signaling inhibitor, was applied. Additionally, Wnt/β-catenin signaling was transiently activated within one day, but the prolonged activation hindered the neuronal differentiation of hUCMSCs. Upon transplantation into the striatum of mice, CiNs survived well and tested positive for dopaminergic neuron markers. They exhibited typical action potentials and sodium and potassium ion channel activity, demonstrating neuronal electrophysiological activity. Furthermore, CiNs treatment significantly increased the number of tyrosine hydroxylase-positive cells and the concentration of dopamine in the striatum, effectively ameliorating movement disorders in PD mice. Overall, our study provides a secure and reliable framework for cell replacement therapy for Parkinson's disease.

Small Molecule Inhibition of APOL1 Channel Activity Protects Podocytes from Mitochondrial Dysfunction, Cell Death, and Barrier Disruption Induced by APOL1 Risk Variants

Small Molecule Inhibition of APOL1 Channel Activity Protects Podocytes from Mitochondrial Dysfunction, Cell Death, and Barrier Disruption Induced by APOL1 Risk Variants

Endosomal fusion of pH-dependent enveloped viruses requires ion channel TRPM7

The majority of viruses classified as pandemic threats are enveloped viruses which enter the cell through receptor-mediated endocytosis and take advantage of endosomal acidification to activate their fusion machinery. Here we report that the endosomal fusion of low pH-requiring viruses is highly dependent on TRPM7, a widely expressed TRP channel that is located on the plasma membrane and in intracellular vesicles. Using several viral infection systems expressing the envelope glycoproteins of various viruses, we find that loss of TRPM7 protects cells from infection by Lassa, LCMV, Ebola, Influenza, MERS, SARS-CoV-1, and SARS-CoV-2. TRPM7 ion channel activity is intrinsically necessary to acidify virus-laden endosomes but is expendable for several other endosomal acidification pathways. We propose a model wherein TRPM7 ion channel activity provides a countercurrent of cations from endosomal lumen to cytosol necessary to sustain the pumping of protons into these virus-laden endosomes. This study demonstrates the possibility of developing a broad-spectrum, TRPM7-targeting antiviral drug to subvert the endosomal fusion of low pH-dependent enveloped viruses.

Decreased ability to manage increases in reactive oxygen species may underlie susceptibility to arrhythmias in mice lacking Scn1b.

Scn1b plays essential roles in the heart, where it encodes β1-subunits that serve as modifiers of gene expression, cell surface channel activity, and cardiac conductivity. Reduced β1 function is linked to electrical instability in various diseases with cardiac manifestations and increased susceptibility to arrhythmias. Recently, we demonstrated that loss of Scn1b in mice leads to compromised mitochondria energetics and reactive oxygen species (ROS) production. In this study, we examined the link between increased ROS and arrhythmia susceptibility in Scn1b-/- mice. In addition, ROS-scavenging capacity can be overwhelmed during prolonged oxidative stress, increasing arrhythmia susceptibility. Therefore, we isolated whole hearts and cardiomyocytes from Scn1b-/- and Scn1b+/+ mice and subjected them to an oxidative challenge with diamide, a glutathione oxidant. Next, we analyzed gene expression and activity of antioxidant enzymes in Scn1b-/- hearts. Cells isolated from Scn1b-/- hearts died faster and displayed higher rates of ROS accumulation preceding cell death compared with those from Scn1b+/+. Furthermore, Scn1b-/- hearts showed higher arrhythmia scores and spent less time free of arrhythmia. Lastly, we found that protein expression and enzymatic activity of glutathione peroxidase is increased in Scn1b-/- hearts compared with wild type. Our results indicate that Scn1b-/- mice have decreased capability to manage ROS during prolonged oxidative stress. ROS accumulation is elevated and appears to overwhelm ROS scavenging through the glutathione system. This imbalance creates the potential for altered cell energetics that may underlie increased susceptibility to arrhythmias or other adverse cardiac outcomes.NEW & NOTEWORTHY Using an oxidative challenge, we demonstrated that isolated cells from Scn1b-/- mice are more susceptible to cell death and surges in reactive oxygen species accumulation. At the whole organ level, they were also more susceptible to the formation of cardiac arrhythmias. This may in part be due to changes to the glutathione antioxidant system.

CNGC20 plays dual roles in regulating plant growth and immunity in Brassica napus

Inflorescence architecture is determined by inflorescence length, branch angles and the density of siliques, which affects planting density, lodging resistance and mechanical operation in rapeseed. However, the molecular mechanisms controlling inflorescence architecture are poorly understood, restricting the progress of breeding varieties with ideal plant architecture in oilseed rape. In this study, we have identified and characterized a rapeseed inflorescence development mutant, reduced inflorescence length (ril), which exhibits determinate and shortened inflorescences, reduced plant height, compact branches, and increased silique density. Through BSA-seq and map-based cloning, we find that RIL encodes a cyclic nucleotide-gated channel 20 (BnaA01.CNGC20). A substitution of proline at the 304th position to leucine (P304L) was identified in the conserved transmembrane domain of BnaA01.CNGC20. This P304L substitution neither affects the subcellular localization and self-assembly of BnaA01.CNGC20, nor disrupts the interactions with BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1), which interacts with CNGC20 and phosphorylates it to regulate Ca2+ channel stability. However, the P304L substitution increases channel activity and Ca2+ influx, which in turn induces immune responses such as cell death, H2O2 accumulation, upregulation of pathogenesis-related genes, and pattern-triggered immunity. The enhanced immunity improves the resistance to Leptosphaeria biglobosa and Sclerotinia sclerotiorum. Transcriptome analysis further revealed that CNGC20 plays dual roles in regulating plant growth and immunity via the brassinosteroid and auxin signaling pathways. The findings in this study provide deeper insights into the intricate relationship between cytosolic Ca2+ level and plant development and immunity, as well as the trade-off between immunity and the performance of yield-related traits in the heterozygous plants (+/ril), which may serve as a guide for balancing yield and disease resistance in oilseed rape breeding.

A Novel De Novo Gain-of-Function CACNA1D Variant in Neurodevelopmental Disease With Congenital Tremor, Seizures, and Hypotonia.

De novo gain-of-function variants in the CACNA1D gene, encoding the L-type voltage-gated Ca2+ channel CaV1.3, cause a multifaceted syndrome. Patients show variable degrees of autism spectrum disorder, developmental delay, epilepsy, and other neurologic and endocrine abnormalities (primary aldosteronism and/or hyperinsulinemic hypoglycemia). We study here a novel variant [c.3506G>A, NM_000720.4, p.(G1169D)] in 2 children with the same CACNA1D mutation but different disease severity. The clinical data of the study patients were collected. After molecular analysis and cloning by site-directed mutagenesis, patch-clamp recordings of transfected tsA201 cells were conducted in whole-cell configuration. The functional effects of wild-type and mutated channels were analyzed. One child is a severely affected boy with a novel de novo CACNA1D variant with additional clinical symptoms including prenatal-onset tremor, congenital respiratory insufficiency requiring continuous positive airway pressure ventilation, and sensorineural deafness. Despite episodes of hypoglycemia, insulin levels were normal. Aldosterone:renin ratios as a screening parameter for primary aldosteronism were variable. In the second patient, putative mosaicism of the p.(G1169D) variant was associated with a less severe phenotype. Patch-clamp electrophysiology of the p.(G1169D) variant in a heterologous expression system revealed pronounced activity-enhancing gating changes, including a shift of channel activation and inactivation to more hyperpolarized potentials, as well as impaired channel inactivation and deactivation. Despite retained sensitivity to the Ca2+ channel blocker isradipine in vitro, no beneficial effects of isradipine or nifedipine treatment were observed in the index case. Through this report, we expand the knowledge about the disease presentation in patients with CACNA1D variants and show the novel variant's modulatory effects on CaV1.3 gating.