PI3K block restores age-dependent neurovascular coupling defects associated with cerebral small vessel disease.

Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) in brain capillary endothelial cells, leading to the loss of inwardly rectifying K+ (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP2 by converting it to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD.

In Vivo Optical Interrogation of Neuronal Responses to Genetic, Cell Type-Specific Silencing.

We established a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expression in vivo Coexpression of a constitutive, Cre-dependent fluorescent marker selectively allowed single-cell analyses before and after inducible, Tet-dependent transgene expression. Here, we used this method for precise, acute manipulation of neuronal activity in the living brain. The goal was to study neuronal network homeostasis at cellular resolution. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least 3 d. Longitudinal in vivo imaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, HighFive) method thus allows visualizing temporally precise, genetic perturbations of defined cells.SIGNIFICANCE STATEMENT Gene function is studied by KO or overexpression of a specific gene followed by analyses of phenotypic changes. However, being able to predict and analyze exactly those cells in which genetic manipulation will occur is not possible. We combined two prominent transgene overexpression methods to fluorescently highlight the targeted cells appropriately before cell type-specific transgene induction. By inducing a potassium channel that decreases neuronal firing, we investigated how neuronal networks in the living mouse brain possibly compensate swift changes in cellular activities. Unlike in vitro, known compensatory homeostatic mechanisms, such as changes in synapses, were not observed in vivo Overall, we demonstrated with our method rapid genetic manipulation and analysis of neuronal activities as well as precision transgene expression.

Visceral adipose of obese mice inhibits endothelial inwardly rectifying K+ channels in a CD36-dependent fashion.

Obesity imposes deficits on adipose tissue and vascular endothelium, yet the role that distinct adipose depots play in mediating endothelial dysfunction in local arteries remains unresolved. We recently showed that obesity impairs endothelial Kir2.1 channels, mediators of nitric oxide production, in arteries of visceral adipose tissue (VAT), while Kir2.1 function in subcutaneous adipose tissue (SAT) endothelium remains intact. Therefore, we determined if VAT versus SAT from lean or diet-induced obese mice affected Kir2.1 channel function in vitro. We found that VAT from obese mice reduces Kir2.1 function without altering channel expression whereas AT from lean mice and SAT from obese mice had no effect on Kir2.1 function as compared to untreated control cells. As Kir2.1 is well known to be inhibited by fatty acid derivatives and obesity is strongly associated with elevated circulating fatty acids, we next tested the role of the fatty acid translocase CD36 in mediating VAT-induced Kir2.1 dysfunction. We found that the downregulation of CD36 restored Kir2.1 currents in endothelial cells exposed to VAT from obese mice. In addition, endothelial cells exposed to VAT from obese mice exhibited a significant increase in CD36-mediated fatty acid uptake. The importance of CD36 in obesity-induced endothelial dysfunction of VAT arteries was further supported in ex vivo pressure myography studies where CD36 ablation rescued the endothelium-dependent response to flow via restoring Kir2.1 and endothelial nitric oxide synthase function. These findings provide new insight into the role of VAT in mediating obesity-induced endothelial dysfunction and suggest a novel role for CD36 as a mediator of endothelial Kir2.1 impairment.NEW & NOTEWORTHY Our findings suggest a role for visceral adipose tissue (VAT) in the dysfunction of endothelial Kir2.1 in obesity. We further reveal a role for CD36 as a major contributor to VAT-mediated Kir2.1 and endothelial dysfunction, suggesting that CD36 offers a potential target for preventing the early development of obesity-associated cardiovascular disease.

Intrauterine exposure to nicotine through maternal vaping disrupts embryonic lung and skeletal development via the Kcnj2 potassium channel.

Smoking cigarettes during pregnancy is associated with adverse effects on infants including low birth weight, defective lung development, and skeletal abnormalities. Pregnant women are increasingly turning to vaping [use of electronic (e)-cigarettes] as a perceived safer alternative to cigarettes. However, nicotine disrupts fetal development, suggesting that like cigarette smoking, nicotine vaping may be detrimental to the fetus. To test the impact of maternal vaping on fetal lung and skeletal development in mice, pregnant dams were exposed to e-cigarette vapor throughout gestation. At embryonic day (E)18.5, vape exposed litter sizes were reduced, and some embryos exhibited growth restriction compared to air exposed controls. Fetal lungs were collected for histology and whole transcriptome sequencing. Maternally nicotine vaped embryos exhibited histological and transcriptional changes consistent with impaired distal lung development. Embryonic lung gene expression changes mimicked transcriptional changes observed in adult mouse lungs exposed to cigarette smoke, suggesting that the developmental defects may be due to direct nicotine exposure. Fetal skeletons were analyzed for craniofacial and long bone lengths. Nicotine directly binds and inhibits the Kcnj2 potassium channel which is important for bone development. The length of the maxilla, palatal shelves, humerus, and femur were reduced in vaped embryos, which was further exacerbated by loss of one copy of the Kcnj2 gene. Nicotine vapor exposed Kcnj2KO/+ embryos also had significantly lower birth weights than unexposed animals of either genotype. Kcnj2 mutants had severely defective lungs with and without vape exposure, suggesting that potassium channels may be broadly involved in mediating the detrimental developmental effects of nicotine vaping. These data indicate that intrauterine nicotine exposure disrupts fetal lung and skeletal development likely through inhibition of Kcnj2.

Potassium channel modulation in macrophages sensitizes dorsal root ganglion neurons after nerve injury.

Macrophages and satellite glial cells are found between injured and uninjured neurons in the lumbar dorsal root ganglia (DRG). We explored the mechanism of neuro-immune and neuron-glia crosstalk leading to hyperexcitability of DRG neurons. After spared nerve injury (SNI), CX3CR1+ resident macrophages became activated, proliferated, and increased inward-rectifying potassium channel Kir 2.1 currents. Conditioned medium (CM) by macrophages, obtained from DRG of SNI mice, sensitized small DRG neurons from naïve mice. However, treatment with CM from GFAP+ glial cells did not affect neuronal excitability. When subjected to this macrophage-derived CM, DRG neurons had increased spontaneous activity, current-evoked responses and voltage-gated NaV 1.7 and NaV 1.8 currents. Silencing Kir 2.1 in macrophages after SNI prevented the induction of neuronal hyperexcitability from their CM. Blocking vesicular exocytosis or soluble tumor necrosis factor in CM or interfering with the downstream intracellular p38 pathway in neurons, also prevented neuronal hyperexcitability. Blocking protein trafficking in neurons reduced the effect of CM, suggesting that the hyperexcitable state resulted from changes in NaV channel trafficking. These results suggest that DRG macrophages, primed by peripheral nerve injury, contribute to neuron-glia crosstalk, NaV channel dysregulation and neuronal hyperexcitability implicated in the development of neuropathic pain.

Hypothalamic POMC neuron-specific knockout of MC4R affects insulin sensitivity by regulating Kir2.1.

BACKGROUND: Imbalance in energy regulation is a major cause of insulin resistance and diabetes. Melanocortin-4 receptor (MC4R) signaling at specific sites in the central nervous system has synergistic but non-overlapping functions. However, the mechanism by which MC4R in the arcuate nucleus (ARC) region regulates energy balance and insulin resistance remains unclear. METHODS: The MC4Rflox/flox mice with proopiomelanocortin (POMC) -Cre mice were crossed to generate the POMC-MC4Rflox/+ mice. Then POMC-MC4Rflox/+ mice were further mated with MC4Rflox/flox mice to generate the POMC-MC4Rflox/flox mice in which MC4R is selectively deleted in POMC neurons. Bilateral injections of 200 nl of AAV-sh-Kir2.1 (AAV-sh-NC was used as control) were made into the ARC of the hypothalamus. Oxygen consumption, carbon dioxide production, respiratory exchange ratio and energy expenditure were measured by using the CLAMS; Total, visceral and subcutaneous fat was analyzed using micro-CT. Co-immunoprecipitation assays (Co-IP) were used to analyze the interaction between MC4R and Kir2.1 in GT1-7 cells. RESULTS: POMC neuron-specific ablation of MC4R in the ARC region promoted food intake, impaired energy expenditure, leading to increased weight gain and impaired systemic glucose homeostasis. Additionally, MC4R ablation reduced the activation of POMC neuron, and is not tissue-specific for peripheral regulation, suggesting the importance of its central regulation. Mechanistically, sequencing analysis and Co-IP assay demonstrated a direct interaction of MC4R with Kir2.1. Knockdown of Kir2.1 in POMC neuron-specific ablation of MC4R restored the effect of MC4R ablation on energy expenditure and systemic glucose homeostasis, indicating by reduced body weight and ameliorated insulin resistance. CONCLUSION: Hypothalamic POMC neuron-specific knockout of MC4R affects energy balance and insulin sensitivity by regulating Kir2.1. Kir2.1 represents a new target and pathway that could be targeted in obesity.

Kir2.1 channel regulates macrophage polarization via the Ca2+/CaMK II/ERK/NF-κB signaling pathway.

Macrophage polarization plays a key role in the inflammatory response. Various ion channels expressed in macrophages have been documented, but very little is known about their roles in macrophage polarization. We found that knockdown or blockade of the Kir2.1 (also known as KCNJ2) channel significantly inhibited M1 macrophage polarization, but promoted M2 macrophage polarization. Lipopolysaccharide (LPS)-induced M1 polarization was also remarkably suppressed in high extracellular K+ solutions (70 mM K+), and this inhibition was partially abolished by adding Ca2+ to the culture medium. Ca2+ imaging showed that Ca2+ influx was dependent on the hyperpolarized membrane potential generated by the Kir2.1 channel. The upregulation of phospho (p)-CaMK II, p-ERK, and p-NF-κB proteins in macrophages from the RAW264.7 cell line that were stimulated with LPS was significantly reversed by blocking the Kir2.1 channel or culturing the cells with 70 mM K+ medium. Furthermore, in vivo studies showed that mice treated with a Kir2.1 channel blocker were protected from LPS-induced peritonitis. In summary, our data reveal the essential role of the Kir2.1 channel in regulating macrophage polarization via the Ca2+/CaMK II/ERK/NF-κB signaling pathway.

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 NO. We previously discovered that flow-induced NO production in resistance arteries of mice and humans critically depends on endothelial inwardly-rectifying K+ channels (Kir2.1). 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 normotensive (n=19; SBP: 115±27mmHg; DBP: 75.3±5.7mmHg) and hypertensive subjects (n=13; SBP: 146.1±15.2 mmHg; DBP: 94.4±6.9mmHg). We find that FIV is impaired in hypertensive adults as demonstrated by a significant reduction in FIV when compared to the normotensive adults, which is partially attributed to a reduction in Kir2.1-dependent vasodilation. Specifically, we show that pharmacologically inhibiting Kir2.1 or functionally downregulating Kir2.1 with endothelial-specific adenoviral vector 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 SBP and DBP, indicating that Kir2.1 contribution to FIV decreases as blood pressure increases. Furthermore, 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 high pressure-induced suppression of Kir2.1 is an important mechanism underlying endothelial dysfunction in hypertension.

Caveolin-3 and Caveolin-1 Interaction Decreases Channel Dysfunction Due to Caveolin-3 Mutations.

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.

Membrane Cholesterol Interactions with Proteins in Hypercholesterolemia-Induced Endothelial Dysfunction.

PURPOSE OF REVIEW: The goal of this review is to highlight work identifying mechanisms driving hypercholesterolemia-mediated endothelial dysfunction. We specifically focus on cholesterol-protein interactions and address specific questions related to the impact of hypercholesterolemia on cellular cholesterol and vascular endothelial function. We describe key approaches used to determine the effects of cholesterol-protein interactions in mediating endothelial dysfunction under dyslipidemic conditions. RECENT FINDINGS: The benefits of removing the cholesterol surplus on endothelial function in models of hypercholesterolemia is clear. However, specific mechanisms driving cholesterol-induced endothelial dysfunction need to be determined. In this review, we detail the latest findings describing cholesterol-mediated endothelial dysfunction, highlighting our studies indicating that cholesterol suppresses endothelial Kir2.1 channels as a major underlying mechanism. The findings detailed in this review support the targeting of cholesterol-induced suppression of proteins in restoring endothelial function in dyslipidemic conditions. The identification of similar mechanisms regarding other cholesterol-endothelial protein interactions is warranted.

Surface translocation of Kir2.1 channel induces IL-1ß secretion in microglia.

One of the major properties of microglia is to secrete cytokines as a reaction to stress such as lipopolysaccharide (LPS) application. The mechanism of cytokine secretion from the microglia upon stress through the inflammasome-mediated release process is well studied, and the voltage-gated Kv1.3 channel is known to play an important role in this process. Most previous studies investigated long-term inflammasome-mediated cytokine release (at least over 4 h) and there are only a few studies on the acute reaction (within minutes order) of the microglia to stress and its cytokine secretion capacity. In this study, we found that LPS induced an increase in Kir2.1 current within 15 min after administration but had no effect on voltage-dependent outward currents. Moreover, cytological and western blot analysis revealed that the increase in the Kir2.1 channel current after LPS administration was induced by the translocation of Kir2.1 from the cytoplasm to the cell surface. From an experiment using the inhibitor and trafficking mutation of Kir2.1, an increase in Kir2.1 was found to contribute to the secretion of the inflammatory cytokine, IL-1ß. Although the physiological significance of this acute IL-1ß secretion remains unclear, our present data imply that Kir2.1 translocation functions as a regulator of IL-1ß secretion, and therefore becomes a potential target to control cytokine release from microglia.

Activation of Kir2.1 improves myocardial fibrosis by inhibiting Ca 2+ overload and the TGF-ß1/Smad signaling pathway.

The inwardly rectifying potassium channel Kir2.1 is closely associated with many cardiovascular diseases. However, the effect and mechanism of Kir2.1 in diabetic cardiomyopathy remain unclear. In vivo, we use STZ to establish the model, and ventricular structural changes, myocardial inflammatory infiltration, and myocardial fibrosis severity are detected by echocardiography, histological staining, immunohistochemistry, and western blot analysis, respectively. In vitro, a myocardial fibrosis model is established with high glucose. The Kir2.1 current amplitude, intracellular calcium concentration, fibrosis-related proteins, and TGF-ß1/Smad pathway proteins are detected by whole-cell patch clamp, calcium probes, western blot analysis, and immunofluorescence, respectively. The in vivo results show that compared to diabetic cardiomyopathy, zacopride (a Kir2.1 selective agonist) significantly reduces the left ventricular systolic diameter and diastolic diameter, increases the left ventricular ejection fraction and left ventricular short-axis shortening, improves the degree of cell necrosis, and reduces the expression of myocardial interstitial fibrosis protein and collagen fibre deposition area. The in vitro results show that the current amplitude and protein expression of Kir2.1 are both decreased in the high glucose-induced myocardial fibrosis model. Additionally, zacopride significantly upregulates the expression of Kir2.1 and inhibits the expressions of the fibrosis-related proteins α-SMA, collagen I, and collagen III. Activation of Kir2.1 reduces the intracellular calcium concentration and inhibits the protein expressions of TGF-ß1 and p-Smad 2/3. Activation of Kir2.1 can improve myocardial fibrosis induced by diabetic cardiomyopathy, and the possible mechanism may be related to inhibiting Ca 2+ overload and the TGF-ß1/Smad signaling pathway.

Differential effects of obesity on visceral versus subcutaneous adipose arteries: role of shear-activated Kir2.1 and alterations to the glycocalyx.

Obesity imposes well-established deficits to endothelial function. We recently showed that obesity-induced endothelial dysfunction was mediated by disruption of the glycocalyx and a loss of Kir channel flow sensitivity. However, obesity-induced endothelial dysfunction is not observed in all vascular beds: visceral adipose arteries (VAAs), but not subcutaneous adipose arteries (SAAs), exhibit endothelial dysfunction. To determine whether differences in SAA versus VAA endothelial function observed in obesity are attributed to differential impairment of Kir channels and alterations to the glycocalyx, mice were fed a normal rodent diet, or a high-fat Western diet to induce obesity. Flow-induced vasodilation (FIV) was measured ex vivo. Functional downregulation of endothelial Kir2.1 was accomplished by transducing adipose arteries from mice and obese humans with adenovirus containing a dominant-negative Kir2.1 construct. Kir function was tested in freshly isolated endothelial cells seeded in a flow chamber for electrophysiological recordings under fluid shear. Atomic force microscopy was used to assess biophysical properties of the glycocalyx. Endothelial dysfunction was observed in VAAs of obese mice and humans. Downregulating Kir2.1 blunted FIV in SAAs, but had no effect on VAAs, from obese mice and humans. Obesity abolished Kir shear sensitivity in VAA endothelial cells and significantly altered the VAA glycocalyx. In contrast, Kir shear sensitivity was observed in SAA endothelial cells from obese mice and effects on SAA glycocalyx were less pronounced. We reveal distinct differences in Kir function and alterations to the glycocalyx that we propose contribute to the dichotomy in SAA versus VAA endothelial function with obesity.NEW & NOTEWORTHY We identified a role for endothelial Kir2.1 in the differences observed in VAA versus SAA endothelial function with obesity. The endothelial glycocalyx, a regulator of Kir activation by shear, is unequally perturbed in VAAs as compared with SAAs, which we propose results in a near complete loss of VAA endothelial Kir shear sensitivity and endothelial dysfunction. We propose that these differences underly the preserved endothelial function of SAA in obese mice and humans.

α1 -Adrenoceptors activate the NLRP3 inflammasome through downregulation of Kir2.1 in cardiac inflammation.

NEW FINDINGS: What is the central question of this study? What is the mechanism of cardiac inflammation induced by α1 -adrenoceptor stimulation by NLRP3 inflammasome activation? What is the main finding and its importance? In the mechanism of cardiac inflammation induced by α1 -adrenoceptor overactivation, Kir2.1 exerts cardioprotective and anti-inflammatory effects by inhibiting the activation of the NLRP3 inflammasome. ABSTRACT: Overstimulation of sympathetic nerves in cardiovascular diseases can lead to impaired cardiomyocyte function and potential heart failure, which activates not only the ß-adrenoceptors but also the α1 -adrenoceptors (α1 -AR). A previous report indicated that NLRP3 inflammasome activation is involved in cardiac inflammation induced by the α1 -AR agonist phenylephrine (PE), but the mechanism is still unknown. Here, we aimed to study whether Kir2.1 is involved in cardiac inflammation caused by PE. The results from in vitro experiments showed that PE upregulated the expression levels of NLRP3, caspase-1, interleukin (IL)-18 and IL-1ß and downregulated the expression level of Kir2.1 in H9C2 cells. The Kir2.1 agonist zacopride downregulated the expression of NLRP3, caspase-1, IL-1ß and IL-18, and the Kir2.1 inhibitor ML133 upregulated their expression. To further explore the mechanism, we found that zacopride downregulated the protein expression level of p-p65 and that ML133 upregulated it. Moreover, the nuclear factor-κB (NF-κB) signalling pathway inhibitor curcumenol reversed the expression of NLRP3 inflammasomes caused by PE in H9C2 cells. In in vivo experiments, the protein expression level of Kir2.1 in the PE group was significantly decreased, and the activation of Kir2.1 by zacopride reduced cardiac inflammation. In short, Kir2.1 is involved in α1 -AR overactivation, which induces cardiac inflammation, through the NF-κB signalling pathway, and activating Kir2.1 can downregulate NLRP3 inflammation and exert cardioprotective effects induced by zacopride.

Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents.

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1WTversus Kir2.1Δ314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.

Hypoxic and osmotic expression of Kir2.1 potassium channels in retinal pigment epithelial cells: Contribution to vascular endothelial growth factor expression.

Retinal pigment epithelial (RPE) cells express different subtypes of inwardly rectifying potassium (Kir) channels. We investigated whether human and rat RPE cells express genes of strongly rectifying Kir2 channels. We also determined the hypoxic and hyperosmotic regulation of Kir2.1 gene expression in cultured human RPE cells and the effects of siRNA-mediated knockdown of Kir2.1 on VEGFA expression, VEGF secretion, proliferation, and viability of the cells. Extracellular hyperosmolarity was induced by addition of NaCl or sucrose. Hypoxia and chemical hypoxia were produced by cell culture in 0.25% O2 and addition of CoCl2, respectively. Gene expression levels were evaluated by real-time RT-PCR. Rat RPE cells contained Kir2.1, Kir2.2, Kir2.3, and Kir2.4 gene transcripts while human RPE cells contained Kir2.1, Kir2.2, and Kir2.4 transcripts. Immunocytochemical data may suggest that Kir2.1 protein in cultured human cells is expressed in both perinuclear and plasma membranes. Kir2.1 gene expression and Kir2.1 protein level in human cells increased under hypoxic and hyperosmotic conditions. The expression of the Kir2.1 gene was mediated in part by diverse intracellular signal transduction pathways and transcription factor activities under both conditions; the hyperosmotic, but not the CoCl2-induced Kir2.1 gene expression was dependent on intracellular calcium signaling. Autocrine/paracrine activation of purinergic receptors contributed to Kir2.1 gene expression under hyperosmotic (P2Y1, P2Y2, P2X7) and CoCl2-induced conditions (P2Y2, P2X7). Exogenous VEGF, TGF-ß1, and blood serum decreased Kir2.1 gene expression. Inhibition of VEGF receptor-2 increased the Kir2.1 gene expression under control conditions and in CoCl2-simulated hypoxia, and decreased it under high NaCl conditions. Knockdown of Kir2.1 by siRNA inhibited the CoCl2-induced and hyperosmotic transcription of the VEGFA gene and caused a delayed decrease of the constitutive VEGFA gene expression while VEGF protein secretion was not altered. Kir2.1 knockdown stimulated RPE cell proliferation under control and hyperosmotic conditions without affecting cell viability. The data indicate that Kir2.1 channel activity is required for the expression of the VEGFA gene and inhibits the proliferation of RPE cells. Under control and hypoxic conditions, the extracellular VEGF level may regulate the production of VEGF via its inhibitory effect on the Kir2.1 gene transcription; this feedback loop may prevent overproduction of VEGF.

Prolonged duration of repolarization and decreased conduction velocity in the atrial myocardium after hypothermic ischemia-reperfusion may be related to expressions of inward rectifier potassium channel 2.1 protein and connexin 40.

OBJECTIVES: The study aimed to determine the role of inward rectifier potassium channel 2.1 protein and connexin 40 expressions in regulating the duration of repolarization and conduction velocity of right atrial myocardium in rats following hypothermic ischemia-reperfusion. METHODS: The Langendorff isolated rat cardiac perfusion models were divided into control (C) and hypothermic ischemia-reperfusion groups, with 8 models in group C and 16 models in group ischemia-reperfusion. Depending on the incidence of atrial arrhythmia after reperfusion, the models in group ischemia-reperfusion were further divided into reperfusion non-atrial arrhythmia or reperfusion atrial arrhythmia subgroup. Right atrial monophasic action potential duration at 50% and 90% of repolarization after 30 minutes of continuous perfusion in group C and group ischemia-reperfusion (T0), 105 minutes of continuous perfusion in group C or after 15 minutes of reperfusion in group ischemia-reperfusion (T1) and 120 minutes of continuous perfusion in group C or 30 minutes of reperfusion in group ischemia-reperfusion (T2) were recorded. Right atrial conduction velocity and effective refractory period were recorded at T2. Then, the expressions of inward rectifier potassium channel 2.1 protein and connexin 40 in the right atrial myocardium were detected. RESULTS: Monophasic action potential duration at 50% and 90% were higher at T1 and T2 than those at T0 in subgroup reperfusion atrial arrhythmia (p < 0.05); monophasic action potential duration at 50% in subgroup reperfusion atrial arrhythmia were larger than group C and subgroup reperfusion non-atrial arrhythmia at T1 and T2 (p < 0.05); monophasic action potential duration at 90% in subgroup reperfusion atrial arrhythmia were larger than group C and subgroup reperfusion non-atrial arrhythmia at T1 and T2 (p < 0.05); effective refractory period in subgroup reperfusion atrial arrhythmia was greater than that in group C and subgroup reperfusion non-atrial arrhythmia, and the conduction velocity and the expressions of inward rectifier potassium channel 2.1 protein and connexin 40 were significantly lower than group C and subgroup reperfusion non-atrial arrhythmia (p < 0.05). CONCLUSIONS: The prolonged duration of repolarization and a decrease in conduction velocity of the atrial myocardium occur in rats after hypothermic ischemia-reperfusion. These observed effects may be related to the downregulated expressions of connexin 40 and inward rectifier potassium channel 2.1.

Up-regulation of miR-195 contributes to cardiac hypertrophy-induced arrhythmia by targeting calcium and potassium channels.

Previous studies have confirmed that miR-195 expression is increased in cardiac hypertrophy, and the bioinformatics website predicted by Targetscan software shows that miR-195 can directly target CACNB1, KCNJ2 and KCND3 to regulate Cavß1, Kir2.1 and Kv4.3 proteins expression. The purpose of this study is to confirm the role of miR-195 in arrhythmia caused by cardiac hypertrophy. The protein levels of Cavß1, Kir2.1 and Kv4.3 in myocardium of HF mice were decreased. After miR-195 was overexpressed in neonatal mice cardiomyocytes, the expression of ANP, BNP and ß-MHC was up-regulated, and miR-195 inhibitor reversed this phenomenon. Overexpression of miR-195 reduced the estimated cardiac function of EF% and FS% in wild-type (WT) mice. Transmission electron microscopy showed that the ultrastructure of cardiac tissues was damaged after miR-195 overexpression by lentivirus in mice. miR-195 overexpression increased the likelihood of arrhythmia induction and duration of arrhythmia in WT mice. Lenti-miR-195 inhibitor carried by lentivirus can reverse the decreased EF% and FS%, the increased incidence of arrhythmia and prolonged duration of arrhythmia induced by TAC in mice. After miR-195 treatment, the protein expressions of Cavß1, Kir2.1 and Kv4.3 were decreased in mice. The results were consistent at animal and cellular levels, respectively. Luciferase assay results showed that miR-195 may directly target CACNB1, KCNJ2 and KCND3 to regulate the expression of Cavß1, Kir2.1 and Kv4.3 proteins. MiR-195 is involved in arrhythmia caused by cardiac hypertrophy by inhibiting Cavß1, Kir2.1 and Kv4.3.

The inhibition of Kir2.1 potassium channels depolarizes spinal microglial cells, reduces their proliferation, and attenuates neuropathic pain.

Spinal microglia change their phenotype and proliferate after nerve injury, contributing to neuropathic pain. For the first time, we have characterized the electrophysiological properties of microglia and the potential role of microglial potassium channels in the spared nerve injury (SNI) model of neuropathic pain. We observed a strong increase of inward currents restricted at 2 days after injury associated with hyperpolarization of the resting membrane potential (RMP) in microglial cells compared to later time-points and naive animals. We identified pharmacologically and genetically the current as being mediated by Kir2.1 ion channels whose expression at the cell membrane is increased 2 days after SNI. The inhibition of Kir2.1 with ML133 and siRNA reversed the RMP hyperpolarization and strongly reduced the currents of microglial cells 2 days after SNI. These electrophysiological changes occurred coincidentally to the peak of microglial proliferation following nerve injury. In vitro, ML133 drastically reduced the proliferation of BV2 microglial cell line after both 2 and 4 days in culture. In vivo, the intrathecal injection of ML133 significantly attenuated the proliferation of microglia and neuropathic pain behaviors after nerve injury. In summary, our data implicate Kir2.1-mediated microglial proliferation as an important therapeutic target in neuropathic pain.

Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4-mediated calcium entry in microglia.

Microglia-mediated inflammation exerts adverse effects in ischemic stroke and in neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the voltage-gated potassium channel Kv1.3 is required for microglia activation. Both genetic deletion and pharmacological inhibition of Kv1.3 are effective in reducing microglia activation and the associated inflammatory responses, as well as in improving neurological outcomes in animal models of AD and ischemic stroke. Here we sought to elucidate the molecular mechanisms underlying the therapeutic effects of Kv1.3 inhibition, which remain incompletely understood. Using a combination of whole-cell voltage-clamp electrophysiology and quantitative PCR (qPCR), we first characterized a stimulus-dependent differential expression pattern for Kv1.3 and P2X4, a major ATP-gated cationic channel, both in vitro and in vivo. We then demonstrated by whole-cell current-clamp experiments that Kv1.3 channels contribute not only to setting the resting membrane potential but also play an important role in counteracting excessive membrane potential changes evoked by depolarizing current injections. Similarly, the presence of Kv1.3 channels renders microglia more resistant to depolarization produced by ATP-mediated P2X4 receptor activation. Inhibiting Kv1.3 channels with ShK-223 completely nullified the ability of Kv1.3 to normalize membrane potential changes, resulting in excessive depolarization and reduced calcium transients through P2X4 receptors. Our report thus links Kv1.3 function to P2X4 receptor-mediated signaling as one of the underlying mechanisms by which Kv1.3 blockade reduces microglia-mediated inflammation. While we could confirm previously reported differences between males and females in microglial P2X4 expression, microglial Kv1.3 expression exhibited no gender differences in vitro or in vivo. MAIN POINTS: The voltage-gated K+ channel Kv1.3 regulates microglial membrane potential. Inhibition of Kv1.3 depolarizes microglia and reduces calcium entry mediated by P2X4 receptors by dissipating the electrochemical driving force for calcium.