Ion Channel Complex Research Articles

Multi-omics inhalation toxicity assessment of urban soil dusts contaminated by multiple legacy sources of lead (Pb)

Although animal studies have evaluated lead (Pb) toxicity, they are limited to soluble forms, such as Pb-acetate, which do not reflect the range found in the exposome. Recent studies on Pb speciation of residential soils in urban areas revealed that the initial Pb sources are not persistent and are extensively repartitioned into adsorbed forms of Pb rather than insoluble phosphates. We investigated the inhalation and neurological toxicity of dusts generated from a surficial soil sample collected from a residential site with an exposomic mixture of various Pb species, both adsorbed phases (Fe and Mn oxide, humate bound Pb) and mineral phases (Pb hydroxycarbonate, pyromorphite, galena). Mice inhaled East Chicago dust (ECD) generated from a composite soil sample for 4 h/day, 7 days/week, for 4 weeks. Mice were necropsied immediately, 1, 14 and 30 days post exposure to evaluate both toxicity and recovery. Exposure to ECD caused changes in memory and spatial learning in the Morris Water Maze test. RNAseq analysis of the hippocampus region revealed multiple differentially expressed genes and impacts on pathways involved in ion channel complexes, and neuron-to-neuron synapse. Metabolomics analysis of plasma highlighted significant alterations in metabolic processes immediately after exposure that resolved after 14 days of rest.

Differential expression of ion channel coding genes in the endometrium of women experiencing recurrent implantation failures

Our study probed the differences in ion channel gene expression in the endometrium of women with Recurrent Implantation Failure (RIF) compared to fertile women. We analyzed the relative expression of genes coding for T-type Ca2+, ENaC, CFTR, and KCNQ1 channels in endometrial samples from 20 RIF-affected and 10 control women, aged 22–35, via microarray analysis and quantitative real-time PCR. Additionally, we examined DNA methylation in the regulatory region of KCNQ1 using ChIP real-time PCR. The bioinformatics component of our research included Gene Ontology analysis, protein–protein interaction networks, and signaling pathway mapping to identify key biological processes and pathways implicated in RIF. This led to the discovery of significant alterations in the expression of ion channel genes in RIF women’s endometrium, most notably an overexpression of CFTR and reduced expression of SCNN1A, SCNN1B, SCNN1G, CACNA1H, and KCNQ1. A higher DNA methylation level of KCNQ1’s regulatory region was also observed in RIF patients. Gene-set enrichment analysis highlighted a significant presence of genes involved with ion transport and membrane potential regulation, particularly in sodium and calcium channel complexes, which are vital for cation movement across cell membranes. Genes were also enriched in broader ion channel and transmembrane transporter complexes, underscoring their potential extensive role in cellular ion homeostasis and signaling. These findings suggest a potential involvement of ion channels in the pathology of implantation failure, offering new insights into the mechanisms behind RIF and possible therapeutic targets.

Cilia-enriched oxysterol 7β,27-DHC is required for polycystin ion channel activation

Polycystin-1 (PC-1) and PC-2 form a heteromeric ion channel complex that is abundantly expressed in primary cilia of renal epithelial cells. This complex functions as a non-selective cation channel, and mutations within the polycystin complex cause autosomal dominant polycystic kidney disease (ADPKD). The spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood. Using both whole-cell and ciliary patch-clamp recordings, we identify a cilia-enriched oxysterol, 7β,27-dihydroxycholesterol (DHC), that serves as a necessary activator of the polycystin complex. We further identify an oxysterol-binding pocket within PC-2 and showed that mutations within this binding pocket disrupt 7β,27-DHC–dependent polycystin activation. Pharmacologic and genetic inhibition of oxysterol synthesis reduces channel activity in primary cilia. In summary, our findings reveal a regulator of the polycystin complex. This oxysterol-binding pocket in PC-2 may provide a specific target for potential ADPKD therapeutics.

A cuproptosis-related gene DLAT as a novel prognostic marker and its relevance to immune infiltration in low-grade gliomas

DLAT has been recognized as a cuproptosis-related gene that is crucial for cuproptosis in earlier research. The study is to look at how DLAT affects individuals with low-grade glioma's prognosis and immune infiltration. The Genotype-Tissue Expression (GTEx) database and the TCGA database were used in this work to download RNAseq data in TPM format. DLAT was found to be overexpressed in LGG by comparing DLAT expression levels between LGG and normal brain tissue, and the expression of DLAT was verified by immunohistochemistry and semi-quantitative analysis. Then, the functional enrichment analysis revealed that the biological functional pathways and possible signal transduction pathways involved were primarily focused on extracellular matrix organization, transmembrane transporter complex, ion channel complex, channel activity, neuroactive ligand-receptor interaction, complement and coagulation cascades, and channel activity. The level of immune cell infiltration by plasmacytoid dendritic cells and CD8 T cells was subsequently evaluated using single-sample gene set enrichment analysis, which showed that high DLAT expression was inversely connected with that level of infiltration. The link between the methylation and mRNA transcription of DLAT was then further investigated via the MethSurv database, and the results showed that DLAT's hypomethylation status was linked to a poor outcome. Finally, by evaluating the prognostic value of DLAT using the Cox regression analysis and Kaplan-Meier technique, a column line graph was created to forecast the overall survival (OS) rate at 1, 3, and 5 years after LGG identification. The aforementioned results demonstrated that high DLAT expression significantly decreased OS and DSS, and that overexpression of DLAT in LGG was significantly linked with WHO grade, IDH status, primary therapy outcome, overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) events. DLAT was discovered as a separate predictive sign of OS in the end. DLAT might thus represent a brand-new predictive biomarker.

Calcium-Dependent Regulation of Neuronal Excitability Is Rescued in Fragile X Syndrome by a Tat-Conjugated N-Terminal Fragment of FMRP.

Fragile X syndrome (FXS) arises from the loss of fragile X messenger ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber long-term potentiation by adjusting the Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knock-out (KO) tsA-201 cells and cerebellar sections from male Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate the A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual-specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signaling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices, cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signaling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of FXS.

Abstract 762: The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts

Abstract Introduction: Colorectal cancer (CRC) is a leading cause of cancer-associated mortality. Our recent study in Pirc rats revealed that the combination of eicosapentaenoic acid and sodium naproxen exhibited additive tumor protection. While the results from this preclinical study were impressive, showing tumor suppression in excess of 95%, potential mechanisms underlying this tumor protection remain unclear. The purpose of this study was to determine the transcriptomic changes resulting in the tumor suppression afforded by the combination of EPA and naproxen supplementation in the diet. Methods: Six week-old Pirc rats were fed either a control AIN-93G diet, or AIN-93G diet supplemented with 2% EPA combined with 200 ppm naproxen, for a total of 20 weeks. Global transcriptomic analysis was performed using RNA sequencing (RNA-Seq) of colon tumors and tumor-adjacent normal tissue from 26-week-old Pirc rats. 100-bp paired-end sequencing was performed on an Illumina NovaSeq to a depth of 25 million reads per sample. Reads were trimmed with Trimmomatic-v0.39, and reads were mapped to the mRatBN7.2.105 rat reference genome using HISAT2. Differentially expressed genes were calculated using DESeq2, and gene set enrichment analysis (GSEA) were performed using Rstudio. Results: DESeq2 identified 809 differentially expressed genes (DEGs, 2-fold change > 1, FDR < 0.05) between large tumors from AIN-93G fed rats versus suppressed tumors from rats fed the combination of EPA and naproxen (490 up-regulated, 319 down-regulated). GSEA analysis revealed activation of inflammatory pathways including IL-17 signaling, JAK-STAT signaling, complement activation, cytokine signaling, ribosome biogenesis, and cell cycle were enriched in untreated control. In contrast, suppressed tumors from animals supplemented with EPA and naproxen were enriched in pathways including negative regulation of Wnt signaling, cell fate specification, ion channel complexes, and nutrient absorption. Furthermore, suppressed tumors shared significant GSEA pathway overlap with normal tissue, leading us to believe the combination of EPA and naproxen may be preventing tumorigenesis in part by maintaining cells in a differentiated state. Conclusion and Future directions: Inflammation plays a key role in tumorigenesis and promoting cancer stem cell niches in tumors. According to our analysis, the combination of EPA and naproxen attenuated cancer-associated inflammation and reduced proliferation in part by augmenting tumor differentiation. Future studies will utilize single-nuclei RNA-Seq (snRNA-Seq) to determine at a single cell resolution the transcriptomic changes of epithelial and stromal populations within the TME. This work was supported by National Cancer Institute task order 75N91019F00132. Citation Format: Ryan Beach, Michael Martinez, Vignesh Gunasekharan, Daniel W. Rosenberg. The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 762.

Postsynaptic β1spectrin maintains Na+ channels at the neuromuscular junction.

Spectrins function together with actin as obligatory subunits of the submembranous cytoskeleton. Spectrins maintain cell shape, resist mechanical forces, and stabilize ion channel and transporter protein complexes through binding to scaffolding proteins. Recently, pathogenic variants of SPTBN4 (β4spectrin) were reported to cause both neuropathy and myopathy. Although the role of β4spectrin in neurons is mostly understood, its function in skeletal muscle, another excitable tissue subject to large forces, is unknown. Here, using a muscle specific β4spectrin conditional knockout mouse, we show that β4spectrin does not contribute to muscle function. In addition, we show β4spectrin is not present in muscle, indicating the previously reported myopathy associated with pathogenic SPTBN4 variants is neurogenic in origin. More broadly, we show that α2, β1 and β2spectrins are found in skeletal muscle, with α2 and β1spectrins being enriched at the postsynaptic neuromuscular junction (NMJ). Surprisingly, using muscle specific conditional knockout mice, we show that loss of α2 and β2spectrins had no effect on muscle health, function or the enrichment of β1spectrin at the NMJ. Muscle specific deletion of β1spectrin also had no effect on muscle health, but, with increasing age, resulted in the loss of clustered NMJ Na+ channels. Together, our results suggest that muscle β1spectrin functions independently of an associated α spectrin to maintain Na+ channel clustering at the postsynaptic NMJ. Furthermore, despite repeated exposure to strong forces and in contrast to neurons, muscles do not require spectrin cytoskeletons to maintain cell shape or integrity. KEY POINTS: The myopathy found in pathogenic human SPTBN4 variants (where SPTBN4 is the gene encoding β4spectrin) is neurogenic in origin. β1spectrin plays essential roles in maintaining the density of neuromuscular junction Nav1.4 Na+ channels. By contrast to the canonical view of spectrin organization and function, we show that β1spectrin can function independently of an associated α spectrin. Despite the large mechanical forces experienced by muscle, we show that spectrins are not required for muscle cell integrity. This is in stark contrast to red blood cells and the axons of neurons.

The structure of the Caenorhabditis elegans TMC-2 complex suggests roles of lipid-mediated subunit contacts in mechanosensory transduction

Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily conserved family of membrane proteins whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila. TMC1 and TMC2 are components of ion channel complexes, but the molecular features that tune these complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here, we present the single-particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans. The complex is composed of two copies of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex highlights conserved protein-lipid interactions, as well as a π-helical structural motif in the pore-forming helices, that together suggest a mechanism for TMC-mediated mechanosensory transduction.

Advanced optical imaging reveals preferred spatial orientation of podocyte processes along the axis of glomerular capillaries

Mammalian kidneys filter enormous volumes of water and small solutes, a filtration driven by the hydrostatic pressure in glomerular capillaries, which is considerably higher than in most other tissues. Interdigitating cellular processes of podocytes form the slits for fluid filtration connected by the membrane-like slit diaphragm cell junction containing a mechanosensitive ion channel complex and allow filtration while counteracting hydrostatic pressure. Several previous publications speculated that podocyte processes may display a preferable orientation on glomerular capillaries instead of a random distribution. However, for decades, the controversy over spatially oriented filtration slits could not be resolved due to technical limitations of imaging technologies. Here, we used advanced high-resolution, three-dimensional microscopy with high data throughput to assess spatial orientation of podocyte processes and filtration slits quantitatively. Filtration-slit-generating secondary processes preferentially align along the capillaries' longitudinal axis while primary processes are preferably perpendicular to the longitudinal direction. This preferential orientation required maturation in development of the mice but was lost in mice with kidney disease due to treatment with nephrotoxic serum or with underlying heterologous mutations in the podocyte foot process protein podocin. Thus, the observation that podocytes maintain a preferred spatial orientation of their processes on glomerular capillaries goes well in line with the role of podocyte foot processes as mechanical buttresses to counteract mechanical forces resulting from pressurized capillaries. Future studies are needed to establish how podocytes establish and maintain their orientation and why orientation is lost under pathological conditions.

Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex.

The slow delayed rectifier potassium current, IKs, conducted through pore-forming Q1 and auxiliary E1 ion channel complexes is important for human cardiac action potential repolarization. During exercise or fright, IKs is up-regulated by protein kinase A (PKA)-mediated Q1 phosphorylation to maintain heart rhythm and optimum cardiac performance. Sympathetic up-regulation of IKs requires recruitment of PKA holoenzyme (two regulatory - RI or RII - and two catalytic Cα subunits) to Q1 C-terminus by an A kinase anchoring protein (AKAP9). Mutations in Q1 or AKAP9 that abolish their functional interaction result in long QT syndrome type 1 and 11, respectively, which increases the risk of sudden cardiac death during exercise. Here, we investigated the utility of a targeted protein phosphorylation (TPP) approach to reconstitute PKA regulation of IKs in the absence of AKAP9. Targeted recruitment of endogenous Cα to E1-YFP using a GFP/YFP nanobody (nano) fused to RIIα enabled acute cAMP-mediated enhancement of IKs, reconstituting physiological regulation of the channel complex. By contrast, nano-mediated tethering of RIIα or Cα to Q1-YFP constitutively inhibited IKs by retaining the channel intracellularly in the endoplasmic reticulum and Golgi. Proteomic analysis revealed that distinct phosphorylation sites are modified by Cα targeted to Q1-YFP compared to free Cα. Thus, functional outcomes of synthetically recruited PKA on IKs regulation is critically dependent on the site of recruitment within the channel complex. The results reveal insights into divergent regulation of IKs by phosphorylation across different spatial and time scales, and suggest a TPP approach to develop new drugs to prevent exercise-induced sudden cardiac death.

A new neurodevelopmental disorder linked to heterozygous variants in UNC79

PurposeThe “NALCN channelosome” is an ion channel complex that consists of multiple proteins, including NALCN, UNC79, UNC80, and FAM155A. Only a small number of individuals with a neurodevelopmental syndrome have been reported with disease causing variants in NALCN and UNC80. However, no pathogenic UNC79 variants have been reported, and in vivo function of UNC79 in humans is largely unknown. MethodsWe used international gene-matching efforts to identify patients harboring ultrarare heterozygous loss-of-function UNC79 variants and no other putative responsible genes. We used genetic manipulations in Drosophila and mice to test potential causal relationships between UNC79 variants and the pathology. ResultsWe found 6 unrelated and affected patients with UNC79 variants. Five patients presented with overlapping neurodevelopmental features, including mild to moderate intellectual disability and a mild developmental delay, whereas a single patient reportedly had normal cognitive and motor development but was diagnosed with epilepsy and autistic features. All displayed behavioral issues and 4 patients had epilepsy. Drosophila with UNC79 knocked down displayed induced seizure-like phenotype. Mice with a heterozygous loss-of-function variant have a developmental delay in body weight compared with wild type. In addition, they have impaired ability in learning and memory. ConclusionOur results demonstrate that heterozygous loss-of-function UNC79 variants are associated with neurologic pathologies.

The Role of RNA Methylation Modification Related Genes in Prognosis and Immunotherapy of Colorectal Cancer.

Researches showed RNA methylation genes can affect the prognosis of tumors. Thus, the study aimed to comprehensively analyze the effects of RNA methylation regulatory genes in prognosis and treatment of colorectal cancer (CRC). Prognostic signature associated with CRCs were constructed by differential expression analysis, Cox and Least Absolute Shrinkage and Selection Operator (LASSO) analyses. Receiver operating characteristic (ROC) and Kaplan-Meier survival analyses were used to validate the reliability of the developed model. Gene Ontology (GO), Gene set variation analysis (GSVA), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for functional annotation. Finally, normal and cancerous tissue were collected to validate gene by quantitative real-time PCR (qRT-PCR). A prognostic risk model based on leucine rich pentatricopeptide repeat containing (LRPPRC) and ubiquitin-like with PHD and ring finger domains 2 (UHRF2) was constructed and relevant to the overall survival (OS) of CRC. Functional enrichment analysis revealed that collagen fibrous tissue, ion channel complex and other pathways were significantly enriched, which might help explain the underlying molecular mechanisms. There were significant differences in ImmuneScore, StromalScore, ESTIMATEScore between high- and low-risk groups (p < 0.05). Ultimately, qRT-PCR validation showed that a significant upregulation in the expression of LRPPRC and UHRF2 in cancerous tissue, which verified the effectiveness of our signature. In conclusion, 2 prognostic genes (LRPPRC and UHRF2) related to RNA methylation were identified by bioinformatics analysis, which might supply a new insight into the treatment and evaluation of CRC.

Structural modeling of peptide toxin-ion channel interactions using RosettaDock.

Voltage-gated ion channels play essential physiological roles in action potential generation and propagation. Peptidic toxins from animal venoms target ion channels and provide useful scaffolds for the rational design of novel channel modulators with enhanced potency and subtype selectivity. Despite recent progress in obtaining experimental structures of peptide toxin-ion channel complexes, structural determination of peptide toxins bound to ion channels in physiologically important states remains challenging. Here we describe an application of RosettaDock approach to the structural modeling of peptide toxins interactions with ion channels. We tested this approach on 10 structures of peptide toxin-ion channel complexes and demonstrated that it can sample near-native structures in all tested cases. Our approach will be useful for improving the understanding of the molecular mechanism of natural peptide toxin modulation of ion channel gating and for the structural modeling of novel peptide-based ion channel modulators.

FacePalm: Probing dynamic S-palmitoylation of CaV channel complexes using engineered depalmitoylases.

S-palmitoylation is a powerful post-translational modification of ion channels that tunes its function and localization. Unlike other lipid modifications, palmitoylation is a reversible process orchestrated by zDHHC family of protein palmitoyl-transferases and acyl-protein thioesterases that attach and excise palmitic groups, respectively. Even so, dissecting spatial and temporal dynamics of palmitoylation of large channel complexes remains challenging, owing to limitations in molecular approaches to acutely manipulate palmitoylation of specific targets. Here, we develop a new approach to rapidly and inducible depalmitoylate target proteins by engineering the ABHD17 (α/β-hydrolase domain containing protein 17) family of protein depalmitoylases with an FKBP/FRBP chemically inducible dimerization system. This approach is termed FKBP/FRB-activated chemogenetic excision of Palmitoylation or FacePalm. Application of FacePalm revealed multifaced regulation of CaV1/2 channel complexes by palmitoylation. First, time-lapse confocal imaging revealed dynamic redistribution of the CaVβ2A subunit from the plasma membrane to the cytoplasm. Second, whole-cell and single-channel electrophysiology show differences in both inactivation and activation of CaV2 channels depending on palmitoylation of distinct sites on either the β2A or the pore-forming α-subunit. Third, targeting FacePalm to distinct subcellular compartments revealed intrinsic differences in the rate of depalmitoylation of CaVβ2A subunit in different organelles. To further demonstrate generality, we applied FacePalm to various cellular proteins and found substrate-specific differences in palmitoylation dynamics. In all, these studies provide critical insights into the exquisite molecular and cellular mechanisms regulating CaV channel function. Furthermore, FacePalm opens new frontiers to dissect dynamic palmitoylation of ion channel complexes in distinct physiological settings.

The role of protein-protein interactions in Fluc dimerization stability and function.

Functional ion channel assembly often requires subunit oligomerization. Yet the molecular factors that define the stability of ion channel complexes in membranes remain to be delineated. Here we investigate this question by studying the dynamic dimerization reaction of the model system Fluc to map out the molecular and physical forces driving this reaction in membranes. Fluc is dual-topology homodimer, where dimerization confers the highly specific fluoride permeation pathway. Our previous analysis demonstrated that a sodium titratable mutant of Fluc participates in a two-step equilibrium dimerization reaction in membranes. First, membrane-dependent dimerization drives subunits to assemble and form a non-contact lipid-solvated stable dimer intermediate by burying a membrane defect imposed in the monomer. However, the fluoride conducting state requires the protein subunits to come closer and expel the lipids to form the fluoride permeation pathway. High resolution crystal structures of Fluc show numerous protein-protein interactions across the dimerization interface, suggesting these interactions might be involved in stabilizing the close-contact dimer state. Alternatively, these contacts may not play a major role in the overall dimer stability but may be important for conferring function. Here, we will investigate how disruption of these protein-protein interactions affects dimerization stability, channel activity or both. Site-directed mutagenesis was employed following four strategies of disruption: (1) reduction of van der Waals interactions by leucine-to-alanine substitutions; (2) removal of potential hydrogen bonds by threonine-to-valine substitutions; (3) decrease backbone flexibility by glycine-to-alanine mutations of the GxxxG dimerization motif; (4) and introduction of bulky tryptophan residues at the interface. Using single-molecule photobleaching analysis to investigate dimer stability in membranes combined with single channel bilayer recordings, these results will provide a quantitative characterization of the contribution of protein-protein interactions to Fluc dimer stability and activity.

A Possible Explanation for the Low Penetrance of Pathogenic KCNE1 Variants in Long QT Syndrome Type 5.

Long QT syndrome (LQTS) is an inherited cardiac rhythm disorder associated with increased incidence of cardiac arrhythmias and sudden death. LQTS type 5 (LQT5) is caused by dominant mutant variants of KCNE1, a regulatory subunit of the voltage-gated ion channels generating the cardiac potassium current IKs. While mutant LQT5 KCNE1 variants are known to inhibit IKs amplitudes in heterologous expression systems, cardiomyocytes from a transgenic rabbit LQT5 model displayed unchanged IKs amplitudes, pointing towards the critical role of additional factors in the development of the LQT5 phenotype in vivo. In this study, we demonstrate that KCNE3, a candidate regulatory subunit of IKs channels minimizes the inhibitory effects of LQT5 KCNE1 variants on IKs amplitudes, while current deactivation is accelerated. Such changes recapitulate IKs properties observed in LQT5 transgenic rabbits. We show that KCNE3 accomplishes this by displacing the KCNE1 subunit within the IKs ion channel complex, as evidenced by a dedicated biophysical assay. These findings depict KCNE3 as an integral part of the IKs channel complex that regulates IKs function in cardiomyocytes and modifies the development of the LQT5 phenotype.

Knockout of the odorant receptor co-receptor, orco, impairs feeding, mating and egg-laying behavior in the fall armyworm Spodoptera frugiperda

The olfactory transduction system of insects is involved in multiple behavioral processes such as foraging, mating, and egg-laying behavior. In the insect olfactory receptor neurons (ORNs), the odorant receptor co-receptor (Orco) is an obligatory component that is required for dimerization with odorant receptors (ORs) to form a ligand-gated ion channel complex. The ORs/Orco heteromeric complex plays a crucial role in insect olfaction. To explore the function of OR-mediated olfaction in the physiological behavior of the fall armyworm, Spodoptera frugiperda, we applied CRISPR/Cas9 genome editing to mutate its Orco gene and constructed a homozygous mutant strain of Orco (Orco−/−) by genetic crosses. Electroantennogram (EAG) analysis showed that the responses of Orco−/− male moths to two universal sex pheromones, Z9-14: Ac and Z7-12: Ac, were abolished. We found that Orco−/− males cannot successfully mate with female moths. An oviposition preference assay confirmed that Orco−/− female moths had a reduced preference for the optimal host plant maize. A larval feeding assay revealed that the time for Orco−/− larvae to locate the food source was significantly longer than in the wild-type. Overall, in the absence of Orco, the OR-dependent olfactory behavior was impaired in both larval and adult stages. Our results confirm that Orco is essential for multiple behavioral processes related to olfaction in the fall armyworm.

Structural basis of Ca2+ uptake by mitochondrial calcium uniporter in mitochondria: a brief review

Mitochondria are cellular organelles that perform various functions within cells. They are responsible for ATP production, cell-signal regulation, autophagy, and cell apoptosis. Because the mitochondrial proteins that perform these functions need Ca2+ ions for their activity, mitochondria have ion channels to selectively uptake Ca2+ ions from the cytoplasm. The ion channel known to play the most important role in the Ca2+ uptake in mitochondria is the mitochondrial calcium uniporter (MCU) holo-complex located in the inner mitochondrial membrane (IMM). This ion channel complex exists in the form of a complex consisting of the pore-forming protein through which the Ca2+ ions are transported into the mitochondrial matrix, and the auxiliary protein involved in regulating the activity of the Ca2+ uptake by the MCU holo-complex. Studies of this MCU holocomplex have long been conducted, but we didn't know in detail how mitochondria uptake Ca2+ ions through this ion channel complex or how the activity of this ion channel complex is regulated. Recently, the protein structure of the MCU holo-complex was identified, enabling the mechanism of Ca2+ uptake and its regulation by the MCU holo-complex to be confirmed. In this review, I will introduce the mechanism of action of the MCU holo-complex at the molecular level based on the Cryo-EM structure of the MCU holo-complex to help understand how mitochondria uptake the necessary Ca2+ ions through the MCU holo-complex and how these Ca2+ uptake mechanisms are regulated. [BMB Reports 2022; 55(11): 528-534].

A prognostic gene signature for gastric cancer and the immune infiltration-associated mechanism underlying the signature gene, PLG.

Globally, gastric cancer (GC) is a common and lethal solid malignant tumor. Identifying the molecular signature and its functions can provide mechanistic insights into GC development and new methods for targeted therapy. Differentially expressed genes (DEGs) and prognostic genes (from univariate Cox regression analysis) were overlapped to obtain prognostic DEGs. Subsequently, molecular modules and the functions of these prognostic DEGs were identified by Metascape and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG)/Gene Set Enrichment Analysis (GSEA) enrichment analyses, respectively. Protein-protein interaction (PPI) networks of up- and down-regulated prognostic DEGs in GC were analyzed using the MCC algorithm of the Cytohubba plug-in in Cytoscape. The prognostic gene signature was defined on hub genes of the PPI networks by least absolute shrinkage and selection operator (LASSO)-Cox regression analysis. Furthermore, the expressional level of PLG in our clinical GC samples was validated by quantitative PCR (qPCR), western blotting, and immunohistochemistry (IHC). Subsequently, the PLG expression-correlation analysis was performed to assess the role of PLG in GC progression. Immune infiltration analysis was performed by single-sample gene set enrichment analysis (ssGSEA) to assess the inhibitory effect of PLG on immune infiltration. Firstly, Up- and down-regulated prognostic DEGs and hub genes in protein-protein interaction (PPI) networks in GC were identified. A prognostic five-gene signature (i.e.,PLG,SPARC,FGB,SERPINE1,andKLHL41) was identified. Among the five genes, the relationship betweenplasminogen(PLG)and GC remains largely unclear. Moreover, the functions of PLG-correlated genes in GC, like 'fibrinolysis', 'hemostasis', 'ion channel complex', and 'transporter complex' were identified. In addition, PLG expression correlated negatively with the infiltration of almost all immune cell types. Interestingly, the expression ofPLGwas significantly and highly correlated with that of CD160, an immune checkpoint inhibitor. Our findings defined a new five-gene signature for predicting GC prognosis, but more validation is required to assess the effects and mechanism of the five genes, especiallyPLG, for the development of new GC therapies.

The potassium channel auxiliary subunit Kvβ2 (Kcnab2) regulates Kv1 channels and dopamine neuron firing.

Ion channel complexes typically consist of both pore-forming subunits and auxiliary subunits that do not directly conduct current but can regulate trafficking or alter channel properties. Isolating the role of these auxiliary subunits in neurons has proved difficult due to a lack of specific pharmacological agents and the potential for developmental compensation in constitutive knockout models. Here, we use cell-type-specific viral-mediated CRISPR/Cas9 mutagenesis to target the potassium channel auxiliary subunit Kvβ2 (Kcnab2) in dopamine neurons in the adult mouse brain. We find that mutagenesis of Kcnab2 reduces surface expression of Kv1.2, the primary Kv1 pore-forming subunit expressed in dopamine neurons, and shifts the voltage dependence of inactivation of potassium channel currents toward more hyperpolarized potentials. Loss of Kcnab2 broadens the action potential waveform in spontaneously firing dopamine neurons recorded in slice, reduces the afterhyperpolarization amplitude, and increases spike timing irregularity and excitability, all of which is consistent with a reduction in potassium channel current. Similar effects were observed with mutagenesis of the pore-forming subunit Kv1.2 (Kcna2). These results identify Kv1 currents as important contributors to dopamine neuron firing and demonstrate a role for Kvβ2 subunits in regulating the trafficking and gating properties of these ion channels. Furthermore, they demonstrate the utility of CRISPR-mediated mutagenesis in the study of previously difficult to isolate ion channel subunits.NEW & NOTEWORTHY Here, we utilize CRISPR/Cas9-mediated mutagenesis in dopamine neurons in mice to target the gene encoding Kvβ2, an auxiliary subunit that forms a part of Kv1 channel complexes. We find that the absence of Kvβ2 alters action potential properties by reducing surface expression of pore-forming subunits and shifting the voltage dependence of channel inactivation. This work establishes a new function for Kvβ2 subunits and Kv1 complexes in regulating dopamine neuron activity.