Explore the vast range of titles available.

Involved in immunity and reproduction, natural killer (NK) cells offer opportunities to develop new immunotherapies to treat infections and cancer or to alleviate pregnancy complications. Most current strategies use cytokines or antibodies to enhance NK-cell function, but none use ion channel modulators, which are widely used in clinical practice to treat hypertension, diabetes, epilepsy, and other conditions. Little is known about ion channels in NK cells. We show that , which codes for the Kir6.1 subunit of a certain type of ATP-sensitive potassium (K ) channel, is highly expressed in murine splenic and uterine NK cells compared to other K channels previously identified in NK cells. expression is highest in the most mature subset of splenic NK cells (CD27 /CD11b ) and in NKG2A or Ly49C/I educated uterine NK cells. Using patch clamping, we show that a subset of NK cells expresses a current sensitive to the Kir6.1 blocker PNU-37883A. does not participate in NK cell degranulation in response to tumor cells in vitro or rejection of tumor cells , or IFN-γ release. Transcriptomics show that genes previously implicated in NK cell development are amongst those differentially expressed in CD27 /CD11b NK cells deficient for . Indeed, we found that mice with NK-cell specific gene ablation have fewer CD27 /CD11b and KLRG-1 NK cells in the bone barrow and spleen. These results show that the K subunit Kir6.1 has a key role in NK-cell development.

Background: K ATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of K ATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various K ATP channel subclasses. Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked “hits”, which were tested for activity against K ATP channels using patch clamping and thallium (Tl + ) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl + fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC 50 of 9 µM and 11 μM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na + currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP 2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.

The 37-kDa laminin receptor (37LRP or RPSA) is a remarkable, multifaceted protein that functions in processes ranging from matrix adhesion to ribosome biogenesis. Its ability to engage extracellular laminin is further thought to contribute to cellular migration and invasion. Most commonly associated with metastatic cancer, RPSA is also increasingly found to be important in other pathologies, including microbial infection, neurodegenerative disease and developmental malformations. Importantly, it is thought to have higher molecular weight forms, including a 67-kDa species (67LR), the expression of which is linked to strong laminin binding and metastatic behavior. The composition of these larger forms has remained elusive and controversial. Homo- and heterodimerization have been proposed as events capable of building the larger species from the monomeric 37-kDa precursor, but solid evidence is lacking. Here, we present data suggesting that higher molecular weight species require SUMOylation to form. We also comment on the difficulty of isolating larger RPSA species for unambiguous identification and demonstrate that cell lines stably expressing tagged RPSA for long periods of time fail to produce tagged higher molecular weight RPSA. It is possible that higher molecular weight species like 67LR are not derived from RPSA.

K channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of K channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various K channel subclasses. We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked \"hits\", which were tested for activity against K channels using patch clamping and thallium (Tl ) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC of 9 µM and 11 μM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.

The leading cause of cardiac death in the United States is arrhythmias, which can result from a variety of reasons. Inherited forms of arrhythmias and sudden unexpected death often result from variants in genes that code for ion channels (or ‘channelopathies’). These arrhythmogenic syndromes can include long-QT syndrome, short-QT syndrome, atrial rhythm disorders, Brugada syndrome and others. Of all the ion channels in the heart, the cardiac Na+ channel, which is responsible for the rapid upstroke of the action potential and fast propagation of the electrical activity through the myocardium, is most often the target. Variants in the SCN5A gene, which encodes the Nav1.5 Na+ channel α-subunit, have been associated with Brugada syndrome (BrS), long QT syndrome (LQTS), sick sinus node syndrome (SSS), atrial fibrillation (AF) and sudden infant death syndrome (SIDS).As part of these studies, we have collaborated with the Office of the Chief Medical Examiner of New York City, who routinely perform genetic testing of ion channel genes in cases of unexplained sudden death. My studies focused on a case of 5‐month‐old girl who died suddenly in her sleep who had heterozygous compound SCN5A variants, leading to an amino acid change (Q1832E) in Nav1.5 and a nonsense mutation that produces a premature stop codon and a C‐terminal truncation (R1944Δ). To investigate their functional relevance, we utilized patch clamp experiments in transfected HEK‐293 cells. The Q1832E mutation drastically reduced Nav1.5 current density, whereas the R1944Δ C‐terminal truncation had negligible effects on Nav1.5 current density. Neither of the mutations affected the voltage dependence of steady state activation and inactivation, the late Na+ current, or the recovery from inactivation. Biochemical and immunofluorescent approaches demonstrated that the Q1832E mutation caused severe trafficking defects. Polymerase chain reaction cloning and sequencing the victim's genomic DNA allowed us to determine that the two variants were in trans. We investigated the functional consequences by co-expressing Nav1.5(Q1832E) and Nav1.5(R1944Δ), which led to a significantly reduced current amplitude relative to wild‐type. These sudden infant death syndrome (SIDS)‐related variants caused a severely dysfunctional Nav1.5 channel, which was mainly due to trafficking defects caused by the Q1832E mutation. Data from this study (and other similar studies that we done) were shared with the OCME, which enhanced their diagnostic capabilities and used for family counseling.We also worked with pediatric cardiologists here at NYU Medical Center, who have identified SCN5A variants in two children with severe arrhythmias. In a family with progressive atrial standstill and sudden death, a novel SCN5A mutation (V1340L) was identified. Patch clamp studies showed that the mutant Nav1.5(V1340L) channel had a reduced current density as well as a depolarizing shift in the voltage-dependent steady-state activation.We additionally investigated a case of an infant with malignant LQT3 and a missense Q1475P SCN5A variant, who was effectively treated with the anti-epileptic drug phenytoin, but the commonly used anti-arrhythmic agent mexiletine was only partially effective. We used a combination of patch clamp and biochemistry studies to show that this variant reduced the current density and channel surface expression, characteristic of a trafficking defect. The mutation also caused a positive shift in the voltage dependence of steady-state activation and inactivation, resulting in a larger “window” current. The rates of inactivation and recovery from inactivation were accelerated, and the “late” Na+ current was increased. We used computational modeling to provide insights into the channel kinetics and to predict effects on the action potential. Simulating channel state transitions with a Markov model demonstrated that a non-inactivated closed state was stabilized by the mutation. Incorporating this model into a simulation of the human ventricular action potential resulted in action potential prolongation. Our patch clamp data showed that mexiletine and phenytoin similarly rescued only some of the gating defects. Surprisingly, mexiletine but not phenytoin, rescued the trafficking defect of the mutant channel, which may explain why it was less effective than phenytoin as an antiarrhythmic drug.Our data make a strong case for the need of functional studies to assess variants of unknown significance in sudden death and arrhythmias. Moreover, there is a need for functional studies before embarking on a one-for-all gene-specific therapy regime of arrhythmias.

•Channelopathies are often associated with inherited arrhythmias and sudden death.•Genetic testing can assist in determining the likely causes of sudden unexpected death.•We identified five SCN10A variants in sudden unexpected death.•Four of the variants led to altered Na+ channel function. Multiple genome-wide association studies (GWAS) and targeted gene sequencing have identified common variants in SCN10A in cases of PR and QRS duration abnormalities, atrial fibrillation and Brugada syndrome. The New York City Office of Chief Medical Examiner has now also identified five SCN10A variants of uncertain significance in six separate cases within a cohort of 330 sudden unexplained death events. The gene product of SCN10A is the Nav1.8 sodium channel. The purpose of this study was to characterize effects of these variants on Nav1.8 channel function to provide better information for the reclassification of these variants. Patch clamp studies were performed to assess effects of the variants on whole-cell Nav1.8 currents. We also performed RNA-seq analysis and immunofluorescence confocal microcopy to determine Nav1.8 expression in heart. We show that four of the five rare ‘variants of unknown significance’ (L388M, L867F, P1102S and V1518I) are associated with altered functional phenotypes. The R756W variant behaved similar to wild-type under our experimental conditions. We failed to detect Nav1.8 protein expression in immunofluorescence microscopy in rat heart. Furthermore, RNA-seq analysis failed to detect full-length SCN10A mRNA transcripts in human ventricle or mouse specialized cardiac conduction system, suggesting that the effect of Nav1.8 on cardiac function is likely to be extra-cardiac in origin. We have demonstrated that four of five SCN10A variants of uncertain significance, identified in unexplained death, have deleterious effects on channel function. These data extend the genetic testing of SUD cases, but significantly more clinical evidence is needed to satisfy the criteria needed to associate these variants with the onset of SUD.

•Channelopathies are often associated with inherited arrhythmias and sudden death.•Genetic testing can assist in determining the likely causes of sudden unexpected death.•We identified four ABCC9 variants in sudden unexpected death.•All four variants caused a gain-of-function phenotype of KATP channels. Genetic variation in ion channel genes (‘channelopathies’) are often associated with inherited arrhythmias and sudden death. Genetic testing (‘molecular autopsies’) of channelopathy genes can be used to assist in determining the likely causes of sudden unexpected death. However, different in silico approaches can yield conflicting pathogenicity predictions and assessing their impact on ion channel function can assist in this regard. We performed genetic testing of cases of sudden expected death in the New York City metropolitan area and found four rare or novel variants in ABCC9, which codes for the regulatory SUR2 subunit of KATP channels. All were missense variants, causing amino acid changes in the protein. Three of the variants (A355S, M941V, and K1379Q) were in cases of infants less than six-months old and one (H1305Y) was in an adult. The predicted pathogenicities of the variants were conflicting. We have introduced these variants into a human SUR2A cDNA, which we coexpressed with the Kir6.2 pore-forming subunit in HEK-293 cells and subjected to patch clamp and biochemical assays. Each of the four variants led to gain-of-function phenotypes. The A355S and M941V variants increased in the overall patch current. The sensitivity of the KATP channels to inhibitory ‘cytosolic’ ATP was repressed for the M941V, H1305Y and K1379Q variants. None of the variants had any effect on the unitary KATP channel current or the surface expression of KATP channels, as determined with biotinylation assays, suggesting that all of the variants led to an enhanced open state. All four variants caused a gain-of-function phenotype. Given the expression of SUR2-containing KATP channels in the heart and specialized cardiac conduction, vascular smooth muscle and respiratory neurons, it is conceivable that electrical silencing of these cells may contribute to the vulnerability element, which is a component of the triple risk model of sudden explained death in infants. The gain-of-function phenotype of these ABCC9 variants should be considered when assessing their potential pathogenicity.

Inherited arrhythmia syndromes are caused by genetic variants that alter cardiac ion channel function. We investigated a complex presentation in a pediatric patient with ventricular tachycardia and conduction abnormalities, harboring a (c.3786G>T) variant, and two inherited variants, (c.2618C>G), and a desmosome (c.1582C>G). The variant, which encodes Cav1.3 L-type calcium channel is the focus of this study, because the C-terminus fragment of Cav1.3 has recently been identified as a transcription auto-enhancer of its own gene and able to prevent arrhythmic events in a mouse model of ischemic heart failure. Leveraging this intrinsic property, we hypothesized that the Cav1.3-C-terminus could reverse the arrhythmic events associated with the variant. Patch-clamp and optical mapping experiments demonstrated a loss of Cav1.3 function, characterized by reduced L-type calcium current densities, and decrease of conduction velocity, leading to inducible re-entrant arrhythmias in human induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs). RNA sequencing confirmed this loss-of-function via the downregulation gene expression. Interestingly, Cav1.3-C-terminus treatment of hiPSC-CMs successfully normalized Cav1.3 gene expression, restored calcium currents, and conduction velocity, and prevented the susceptibility to arrhythmias. These findings highlight the electrophysiological consequences resulting from a Cav1.3 variant and demonstrate an important transcriptional role of Cav1.3-C-terminus as a transcriptional regulator and as a promising therapeutic tool to restore normal electrical properties in patients with calcium channels loss of function.

Involved in immunity and reproduction, natural killer (NK) cells offer opportunities to develop new immunotherapies to treat infections and cancer or to alleviate pregnancy complications. Most current strategies use cytokines or antibodies to enhance NK-cell function, but none use ion channel modulators, which are widely used in clinical practice to treat hypertension, diabetes, epilepsy, and other conditions. Little is known about ion channels in NK cells. We show that which codes for the Kir6.1 subunit of a certain type of ATP-sensitive potassium (K ) channel, is highly expressed in murine splenic and uterine NK cells compared to other K channels previously identified in NK cells. expression is highest in the most mature subset of splenic NK cells (CD27 CD11b ) and in NKG2A or Ly49C/I educated uterine NK cells. Using patch clamping, we show that a subset of NK cells expresses a current sensitive to the Kir6.1 blocker PNU-37883A. does not participate in NK cell degranulation in response to tumor cells or rejection of tumor cells . Transcriptomics show that genes previously implicated in NK cell development are amongst those differentially expressed in CD27 CD11b NK cells deficient of . Indeed, we found that mice with NK-cell specific gene ablation have fewer CD11b CD27 and KLRG-1 NK cells in the bone barrow and spleen. These results show that the K subunit Kir6.1 has a key role in NK-cell development.

Background Jingmen tick virus (JMTV), a segmented orthoflavi-like virus, has been recognized in recent years as a potential human and animal pathogen, with confirmed detections across Asia, Europe, Africa and South America. Recent JMTV surveillance data from Guinea has enabled detailed population genetic analysis to better understand the epidemiology of the virus in West Africa. Here, we report the results of a JMTV genetic diversity study and address specific issues of virus circulation in ticks parasitizing domestic animals. Methods A total of 928 ticks were collected from 114 host animals across 14 Guinean prefectures, and virus isolation was subsequently attempted in the tick Hyalomma anatolicum (HAE/CTVM8 cell line) cell culture. All collected ticks were morphologically identified, and PCR screening for JMTV was performed. Positive samples were subjected to either complete genome sequencing or targeted fragment sequencing. Results Our sample included nine tick species, among which only three were involved in JMTV circulation: Rhipicephalus microplus , Rhipicephalus geigyi and Amblyomma variegatum . A total of 90 tick samples tested positive for JMTV. One JMTV strain was successfully isolated in the HAE/CTVM8 cell culture. Complete genomes (all 4 segments) were sequenced for six isolates to subsequently perform genetic diversity analysis, while a fragment of segment 3 was sequenced for 64 samples to assess genetic diversity and conduct haplotype analysis. We identified and mapped three natural foci and demonstrated potential JMTV circulation in some of these over 3 years. An analysis of the ticks collected from JMTV-exposed hosts showed 40.4% PCR positivity among attached ticks. Phylogenetic and haplotype analyses demonstrated a remarkably low genetic diversity among Guinean isolates, forming a monophyletic cluster with a star-like haplotype network topology; these are patterns consistent with recent viral introduction. We also detected a reassortment event involving segment 3. Conclusions These findings suggest that JMTV has recently been introduced to Guinea through livestock trade networks, with R.   microplus ticks likely facilitating its spread. The evidence of reassortment highlights the adaptive potential of this virus in new ecosystems. Graphical abstract