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Expression, purification, and functional reconstitution of mammalian ion channels are often challenging. Heterologous expression of mammalian channels in bacteria can be advantageous due to unrelated protein environment and the lack of risk of copurification of endogenous proteins, e.g., accessory channel subunits that can influence the channel activity. Also, direct recording of channel activity could be challenging due to their intracellular localization like in the case of mitochondrial channels. The activity of purified channels can be characterized at the single-molecule level by electrophysiological techniques, such as planar lipid bilayers (PLB). In this work, we describe a simple approach to accomplish PLB recording of the activity of single renal outer medullary potassium channels ROMK expressed in E. coli. We focused on the ROMK2 isoform that is present at low levels in the mitochondria and can be responsible for mitoK ATP activity. We screened for the best construct to express the codon-optimized ROMK proteins with a 6xHis tag for protein purification. The strategy involved the use of optimal styrene-maleic acid (SMA) copolymer, which forms so-called polymer nanodiscs, to solubilize and purify ROMK-containing SMA lipid particles (SMALPs), which were amenable for fusion with PLB. Reconstituted ROMK channels exhibited ion selectivity, rectification, and pharmacological properties, which are in agreement with previous work on ROMK channels., (Copyright © 2021. Published by Elsevier B.V.)

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., (© 2020 Wiley Periodicals, Inc.)

Background: Soluble epoxide hydrolase inhibitors (sEHi) have anti-arrhythmic effects, and we previously found that the novel sEHi t-AUCB (trans-4[-4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid) significantly inhibited ventricular arrhythmias after myocardial infarction (MI). However, the mechanism is unknown. It's known that microRNA-29 (miR-29) participates in the occurrence of arrhythmias. In this study, we investigated whether sEHi t-AUCB was protective against ischemic arrhythmias by modulating miR-29 and its target genes KCNJ12 and KCNIP2., Methods: Male 8-week-old C57BL/6 mice were divided into five groups and fed distilled water only or distilled water with t-AUCB of different dosages for seven days. Then, the mice underwent MI or sham surgery. The ischemic region of the myocardium was obtained 24 hours after MI to detect miR-29, KCNJ12, and KCNIP2 mRNA expression levels via real-time PCR and KCNJ12 and KCNIP2 protein expression levels via western blotting., Results: MiR-29 expression levels were significantly increased in the ischemic region of MI mouse hearts and the mRNA and protein expression levels of its target genes KCNJ12 and KCNIP2 were significantly decreased. T-AUCB prevented these changes dose-dependently., Conclusion: The sEHi t-AUCB regulates the expression levels of miR-29 and its target genes KCNJ12 and KCNIP2, suggesting a possible mechanism for its potential therapeutic application in ischemic arrhythmia.

Background: We aimed to clarify the role of potassium voltage-gated channel subfamily J member 15 (KCNJ15) in esophageal squamous cell carcinoma (ESCC) cells and its potential as a prognosticator in ESCC patients., Methods: KCNJ15 transcription levels were evaluated in 13 ESCC cell lines and polymerase chain reaction (PCR) array analysis was conducted to detect coordinately expressed genes with KCNJ15. The biological functions of KCNJ15 in cell invasion, proliferation, migration, and adhesion were validated through small interfering RNA-mediated knockdown experiments. Cell proliferation was further evaluated through the forced expression experiment. KCNJ15 expression was detected in 200 ESCC tissues by quantitative real-time reverse transcription PCR (qRT-PCR) and analyzed in 64 representative tissues by immunohistochemistry. Correlations between KCNJ15 expression levels and clinicopathological features were also analyzed., Results: The KCNJ15 expression levels varied widely in ESCC cell lines and correlated with COL3A1, JAG1, and F11R. Knockdown of KCNJ15 expression significantly repressed cell invasion, proliferation, and migration of ESCC cells in vitro. Furthermore, overexpression of KCNJ15 resulted in increased cell proliferation. Patients were stratified using the cut-off value of KCNJ15 messenger RNA (mRNA) levels in 200 ESCC tissues using receiver operating characteristic curve analysis; the high KCNJ15 expression group had significantly shorter overall and disease-free survival times. In multivariable analysis, high expression of KCNJ15 was identified as an independent poor prognostic factor. Staining intensity of in situ KCNJ15 protein expression tended to be associated with KCNJ15 mRNA expression levels., Conclusions: KCNJ15 is involved in aggressive tumor phenotypes of ESCC cells and its tissue expression levels may be useful as a prognosticator of patients with ESCC.

Purpose: Diabetic retinopathy (DR) is a leading cause of visual impairment. Müller cells in DR are dysfunctional due to downregulation of the inwardly rectifying potassium channel Kir4.1. Metformin, a commonly used oral antidiabetic drug, is known to elicit its action through 5' adenosine monophosphate-activated protein kinase (AMPK), a cellular metabolic regulator; however, its effect on Kir4.1 channels is unknown. For this study, we hypothesized that metformin treatment would correct circadian rhythm disruption and Kir4.1 channel dysfunction in db/db mice., Methods: Metformin was given orally to db/db mice. Wheel-running activity, retinal levels of Kir4.1, and AMPK phosphorylation were determined at study termination. In parallel, rat retinal Müller cell line (rMC-1) cells were treated using metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to assess the effect of AMPK activation on the Kir4.1 channel., Results: The wheel-running activity of the db/db mice was improved following the metformin treatment. The Kir4.1 level in Müller cells was corrected after metformin treatment. Metformin treatment led to an upregulation of clock regulatory genes such as melanopsin (Opn4) and aralkylamine N-acetyltransferase (Aanat). In rMC-1 cells, AMPK activation via AICAR and metformin resulted in increased Kir4.1 and intermediate core clock component Bmal-1 protein expression. The silencing of Prkaa1 (gene for AMPKα1) led to decreased Kir4.1 and Bmal-1 protein expression., Conclusions: Our findings demonstrate that metformin corrects abnormal circadian rhythm and Kir4.1 channels in db/db mouse a model of type 2 diabetes. Metformin could represent a critical pharmacological agent for preventing Müller cell dysfunction observed in human DR.

Purpose: Diabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels., Methods: We assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA., Results: The IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1., Conclusions: Our studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1.

Diversified neurons are essential for sensorimotor function, but whether astrocytes become specialized to optimize circuit performance remains unclear. Large fast α-motor neurons (FαMNs) of spinal cord innervate fast-twitch muscles that generate peak strength. We report that ventral horn astrocytes express the inward-rectifying K + channel Kir4.1 (a.k.a. Kcnj10) around MNs in a VGLUT1-dependent manner. Loss of astrocyte-encoded Kir4.1 selectively altered FαMN size and function and led to reduced peak strength. Overexpression of Kir4.1 in astrocytes was sufficient to increase MN size through activation of the PI3K/mTOR/pS6 pathway. Kir4.1 was downregulated cell autonomously in astrocytes derived from amyotrophic lateral sclerosis (ALS) patients with SOD1 mutation. However, astrocyte Kir4.1 was dispensable for FαMN survival even in the mutant SOD1 background. These findings show that astrocyte Kir4.1 is essential for maintenance of peak strength and suggest that Kir4.1 downregulation might uncouple symptoms of muscle weakness from MN cell death in diseases like ALS., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Basal and acetylcholine-gated inward-rectifier K + -currents (I K1 and I K,ACh , respectively) are altered in atrial fibrillation (AF). G i -protein-coupled muscarinic (M) receptors type-2 are considered the predominant receptors activating I K,ACh . Although a role for G q -coupled non-M 2 -receptor subtypes has been suggested, the precise regulation of I K,ACh by multiple M-receptor subtypes in the human atrium is unknown. Here, we investigated M 1 -receptor-mediated I K,ACh regulation and its remodeling in chronic AF (cAF)., Methods and Results: M 1 -receptor mRNA and protein abundance were increased in atrial cardiomyocyte fractions and atrial homogenates from cAF patients, whereas M 2 -receptor levels were unchanged. The regulation of I K,ACh by M 1 -receptors was investigated in right-atrial cardiomyocytes using two applications of the M-receptor agonist carbachol (CCh, 2μM), with pharmacological interventions during the second application. CCh application produced a rapid current increase (Peak-I K,ACh ), which declined to a quasi-steady-state level (Qss-I K,ACh ). In sinus rhythm (Ctl) the selective M 1 -receptor antagonists pirenzepine (10nM) and muscarinic toxin-7 (MT-7, 10nM) significantly inhibited CCh-activated Peak-I K,ACh , whereas in cAF they significantly reduced both Peak- and Qss-I K,ACh , with no effects on basal inward-rectifier currents in either group. Conversely, the selective M 1 -receptor agonist McN-A-343 (100μM) induced a current similar to the CCh-activated current in Ctl atrial cardiomyocytes pretreated with pertussis toxin to inhibit M 2 -receptor-mediated G i -protein signaling, which was abolished by MT-7. Computational modeling indicated that M 1 - and M 2 -receptors redundantly activate I K,ACh to abbreviate APD, albeit with predominant effects of M 2 -receptors., Conclusion: Our data suggest that G q -coupled M 1 -receptors also regulate human atrial I K,ACh and that their relative contribution to I K,ACh activation is increased in cAF patients. We provide novel insights about the role of non-M 2 -receptors in human atrial cardiomyocytes, which may have important implications for understanding AF pathophysiology., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Diabetic retinopathy (DR) is a major cause of adult blindness. Retinal Müller cells maintain water homeostasis and potassium concentration via inwardly rectifying Kir4.1 channels. Accumulation of advanced glycation end products (AGEs) is a major pathologic event in DR. While diabetes leads to a decrease in the Kir4.1 channels, it remains unknown whether AGEs-linked to the basement membrane (BM) affect normal Kir4.1 channels. For this study, we hypothesized that AGE-modification of laminin is detrimental to Kir4.1 channels, therefore, disrupting Müller cell function. The AGE-modified laminin-coated substrates were prepared by incubating Petri-dishes with laminin and methylglyoxal for seven days. The rat Müller cells (rMC-1) were propagated on AGE-modified laminin, and Kir4.1 expression and function were evaluated. Quantification of AGEs using ELISA revealed a dose-dependent increase in methylglyoxal-hydro-imidazolone adducts. The rMC-1 propagated on AGE-modified laminin demonstrated a decrease in Kir4.1 levels in immunofluorescence and western blot studies and a decrease in the Kir4.1 channel function. Kir4.1 decrease on AGE-modified laminin resulted in a disorganization of an actin cytoskeleton and disruption of α-dystroglycan-syntrophin-dystrophin complexes. Our studies suggest that AGE-modification of laminin is detrimental to Kir4.1 channels. By studying the role of AGEs in Kir4.1 channels we have identified a novel mechanism of Müller cell dysfunction and its subsequent involvement in DR.

Introduction: The left ventricular hypertrophy (LVH)-ventricular arrhythmias relationship associated with arterial hypertension and aging remains controversial. We aimed to assess the age-dependency of ventricular arrhythmias in spontaneously hypertensive rats (SHRs) and the corresponding ventricular structural and molecular remodeling., Materials and Methods: Ventricular arrhythmias were quantified using 24-h radiotelemetry ECG monitoring in eight SHRs and four Wistar-Kyoto (WKY) rats at 14 (young), 24 (adult), and 48 (aging) weeks of age. Left ventricular histology and mRNA expressions of 89 proarrhythmogenic genes were assessed in six additional groups (n=4 each) of young, adult, and aging SHRs and WKYs., Results: Regardless of their age, SHRs presented more premature ventricular contractions (PVCs) than age-matched WKYs (p<0.01). The arrhythmogenicity peak occurred in adult SHRs; ventricular tachycardias only occurred in adult SHRs. Among the SHRs, LV thickness, interstitial fibrosis, and the number of deregulated genes increased with age. Kcnj11 expression was deregulated in adult, but not in young or aging SHRs., Discussion: This study confirms the presence of higher ventricular ectopy in SHRs than in age-matched WKYs. LVH appeared to be an adaptive, antiarrhythmic process. Myocardial energetic changes with advancing age, as reflected by Kcnj11 expression changes, could underlie this age-dependency of ventricular arrhythmias., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: Diabetic patients often are affected by vision problems. We previously identified diabetic retinopathy (DR) as a disease of clock gene dysregulation. TNF-α, a proinflammatory cytokine, is known to be elevated in DR. Müller cells maintain retinal water homeostasis and K+ concentration via Kir4.1 channels. Notably, Kir4.1 expression is reduced in diabetes; however, the interplay of TNF-α, Kir4.1, and clock genes in Müller cells remains unknown. We hypothesize that the Kir4.1 in Müller cells is under clock regulation, and increase in TNF-α is detrimental to Kir4.1., Methods: Long-Evans rats were made diabetic using streptozotocin (STZ). Retinal Kir4.1 expression was determined at different time intervals. Rat Müller (rMC-1) cells were transfected with siRNA for Per2 or Bmal1 and in parallel treated with TNF-α (5-5000 pM) to determine Kir4.1 expression., Results: Kir4.1 expression exhibited a diurnal rhythm in the retina; however, with STZ-induced diabetes, Kir4.1 was reduced overall. Kir4.1 rhythm was maintained in vitro in clock synchronized rMC-1 cells. Clock gene siRNA-treated rMC-1 exhibited a decrease in Kir4.1 expression. TNF-α treatment of rMCs lead to a profound decrease in Kir4.1 due to reduced colocalization of Kir4.1 channels with synapse-associated protein (SAP97) and disorganization of the actin cytoskeleton., Conclusions: Our findings demonstrate that Kir4.1 channels possess a diurnal rhythm, and this rhythm is dampened with diabetes, thereby suggesting that the increase in TNF-α is detrimental to normal Kir4.1 rhythm and expression.

Background: The E23K variant of the Kir6.2 subunit of the ATP-sensitive potassium (KATP) channel has been implicated in cardiac remodeling. However, the effects of E23K variant on ventricular electrophysiology and arrhythmogenesis remain unclear., Methods: Transgenic rats were generated to express human E23K-variant genomic DNA in the heart under the α-myosin heavy chain promoter. Electrophysiological parameters including electrocardiograph, ventricular action potential duration (APD), effective refractory period (ERP), electrical alternans and ventricle arrhythmia threshold were examined in wild type (WT) and transgenic rats. The KATP current in cardiomyocytes was recorded using whole-cell patch clamp techniques., Results: No differences in the electrophysiological parameters between the two groups were found at baseline. However, after acute ischemic stress, shortened QT intervals were further aggravated in the E23K-variant rats. Additionally, the E23K variant exacerbated the decrease of APD 70 , APD 90 and ERP. The ventricular arrhythmia and alternans thresholds were significantly attenuated, and the duration of ventricular arrhythmia induced by electrical stimulation was significantly prolonged in the E23K-variant rats. More importantly, the KATP current in cardiomyocytes was significantly increased in the E23K-variant rats after ischemia., Conclusion: The E23K variant of the KATP channel increased the susceptibility to ventricular arrhythmia under acute ischemia stress., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: To test the effects of rearing light intensity on retinal function and morphology in the retinoschisis knockout (Rs1-KO) mouse model of X-linked retinoschisis, and whether it affects functional outcome of RS1 gene replacement., Methods: Seventy-six Rs1-KO mice were reared in either cyclic low light (LL, 20 lux) or moderate light (ML, 300 lux) and analyzed at 1 and 4 months. Retinal function was assessed by electroretinogram and cavity size by optical coherence tomography. Expression of inward-rectifier K+ channel (Kir4.1), water channel aquaporin-4 (AQP4), and glial fibrillary acidic protein (GFAP) were analyzed by Western blotting. In a separate study, Rs1-KO mice reared in LL (n = 29) or ML (n = 27) received a unilateral intravitreal injection of scAAV8-hRs-IRBP at 21 days, and functional outcome was evaluated at 4 months by electroretinogram., Results: At 1 month, no functional or structural differences were found between LL- or ML-reared Rs1-KO mice. At 4 months, ML-reared Rs1-KO mice showed significant reduction of b-wave amplitude and b-/a-wave ratio with no changes in a-wave, and a significant increase in cavity size, compared to LL-reared animals. Moderate light rearing increased Kir4.1 expression in Rs1-KO mice by 4 months, but not AQP4 and GFAP levels. Administration of scAAV8-hRS1-IRBP to Rs1-KO mice showed similar improvement of inner retinal ERG function independent of LL or ML rearing., Conclusions: Rearing light conditions affect the development of retinal cavities and post-photoreceptor function in Rs1-KO mice. However, the effect of rearing light intensity does not interact with the efficacy of RS1 gene replacement in Rs1-KO mice.

To elucidate the complex molecular mechanisms of anaplastic thyroid carcinoma (ATC), the mRNA and miRNA expression profiles of ATC were systematically explored. A total of 55 common differentially expressed genes (DEGs) were obtained from two mRNA expression datasets including 23 ATC samples and 24 paired normal samples. Gene expression levels of three randomly selected DEGs, VCAN, COL5A1 and KCNJ16, were examined using RT-PCR in 10 ATC samples. Notably, the ATC and normal samples were clearly classified into two groups based on their common DEGs. Moreover 23 common DEGs, such as TG, NKX2-1, KCNJ16 and CTHRC1, were predicted to be the potential targets of 17 identified miRNAs in ATC. Meanwhile, several miRNA target genes were associated with biological processes related to tumor progression such as angiogenesis, cell migration or growth and potassium channel regulation. In summary, the poor prognosis of ATC is possibly caused via complex biological processes. Firstly, angiogenesis was activated by the high expression of CTHRC1, VCAN and POSTN, providing necessary nutrition for tumor cells. Then tumor distant metastasis was induced via stimulation of cell migration and cell growth or regulation of cell-cell interaction. Moreover, intracellular potassium concentration changes promoted ATC progression indirectly. Hence, identification of these critical DEGs was valuable in understanding the molecular mechanisms of ATC.

Kir4.1 is an inwardly rectifying potassium (K(+)) channel and is expressed in the brain, inner ear, and kidney. In the kidney, Kir4.1 is expressed in the basolateral membrane of the late thick ascending limb (TAL), the distal convoluted tubule (DCT), and the connecting tubule (CNT)/cortical collecting duct (CCD). It plays a role in K(+) recycling across the basolateral membrane in corresponding nephron segments and in generating negative membrane potential. The renal phenotypes of the loss-function mutations of Kir4.1 include mild salt wasting, hypomagnesemia, hypokalemia, and metabolic alkalosis, suggesting that the disruption of Kir4.1 mainly impairs the transport in the DCT. Patch-clamp experiments and immunostaining demonstrate that Kir4.1 plays a predominant role in determining the basolateral K(+) conductance in the DCT. However, the function of Kir4.1 in the TAL and CNT/CCD is not essential, because K(+) channels other than Kir4.1 are also expressed. The downregulation of Kir4.1 in the DCT reduced basolateral chloride (Cl(-)) conductance, suppressed the expression of ste20 proline-alanine-rich kinase (SPAK), and decreased Na-Cl cotransporter (NCC) expression and activity. This suggests that Kir4.1 regulates NCC expression by the modulation of the Cl(-)-sensitive with-no-lysine kinase-SPAK pathway., (Copyright © 2016 the American Physiological Society.)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Astroglial inward rectifying Kir4.1 potassium channels are fundamental for the maintenance of ion and water homeostasis in the central nervous system (CNS). Down-regulation of Kir4.1 expression is observed in CNS disorders associated with excessive extracellular glutamate (Glu) accumulation, including hepatic encephalopathy related to acute liver failure (ALF). Here we demonstrate that prolonged (3 days) treatment of cultured rat cortical astrocytes with 2 mM Glu or 100 µM NMDA decreases the expression of Kir4.1 mRNA and protein. Inhibition by Glu of Kir4.1 mRNA expression was reversed by NMDA receptor antagonists MK-801 and AP-5 (each at 50 µM), and by a non-transportable inhibitor of Glu uptake TBOA (100 µM). MK-801 reversed the inhibitory effect of Glu on Kir4.1 protein expression. In contrast, transcription of Kir4.1 channels was not affected by: (i) a transportable Glu uptake inhibitor PDC (100 µM); (ii) by group I mGluR antagonist MTEP (100 µM); (iii) by antagonists of oxidative-nitrosative stress (ONS) in astrocytes, including the neuroprotective amino acid taurine (Tau; 10 mM), the NADPH oxidase inhibitor apocyanine (APO; 300 µM), the nitric oxide synthase inhibitor, L-NNA (100 µM), and a membrane permeable glutathione precursor, glutathione-diethyl ester (GEE; 3 mM). Down-regulation of Kir4.1 transcription in rats with ALF was attenuated by intraperitoneal administration of a competitive NMDA receptor antagonist memantine, but not by histidine, which reverses ONS associated with ALF. Collectively, the results indicate that over-activation of astroglial NMDA receptors, aided by as yet undefined effects of Glu entry to astrocytes, is a primary cause of the reduction of Kir4.1 expression in CNS disorders associated with increased exposure to Glu., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Gastric acid secretion is mediated by the K(+)-dependent proton pump (H(+),K(+)-ATPase), which requires a continuous supply of K(+) at the luminal side of the apical membrane. Several K(+) channels are implicated in gastric acid secretion. However, the identity of the K(+) channel(s) responsible for apical K(+) supply is still elusive. Our previous studies have shown the translocation of KCNJ15 from cytoplasmic vesicles to the apical membrane on stimulation, indicating its involvement in gastric acid secretion. In this study, the stimulation associated trafficking of KCNJ15 was observed in a more native context with a live cell imaging system. KCNJ15 molecules in resting live cells were scattered in cytoplasm but exhibited apical localization after stimulation. Furthermore, knocking down KCNJ15 expression with a short hairpin RNA adenoviral construct abolished histamine-stimulated acid secretion in rabbit primary parietal cells. Moreover, KCNJ15, like H(+),K(+)-ATPase, was detected in all of the parietal cells by immunofluorescence staining, whereas only about half of the parietal cells were positive for KCNQ1 under the same condition. Consistently, the endogenous protein levels of KCNJ15, analyzed by Western blotting, were higher than those of KCNQ1 in the gastric mucosa of human, mouse, and rabbit. These results provide evidence for a major role of KCNJ15 in apical K(+) supply during stimulated acid secretion., (Copyright © 2015 the American Physiological Society.)

Müller cells are principal glial cells in rat retina and have attracted much attention in glaucoma studies. However, it is not clear whether adenosine and adenosine receptor (AR) antagonists play any roles in the regulation of potassium channels in Müller cells and subsequently in the promotion of glutamine synthetase (GS) and L-Glutamate/L-Aspartate Transporter (GLAST) functions. We found that chronic ocular hypertension (COH) in rat down-regulated Müller cells Kir2.1, Kir4.1, TASK-1, GS and GLAST expressions and attenuated the peak of inward potassium current. Retinal ganglion cells (RGC) count was lower in the COH rats than that in the sham operation animals. Intravitreal injection of selective A2A AR antagonist SCH442416 up-regulated Müller cell Kir4.1, TASK-1, GS and GLAST expressions and enhanced inward potassium currents compared with those in the COH rats with vehicle control. Meanwhile, the RGC count was higher following intravitreal injection of SCH442416 in the COH rats than that after vehicle injection. The fact that PKA inhibitor H-89 blocked these SCH442416 effects suggested that the PKA signaling pathway was involved in the observed ocular responses following the intravitreal SCH442416 injection.

Purpose: Myocardial infarction (MI) results in an increased susceptibility to ventricular arrhythmias, due in part to decreased inward-rectifier K+ current (IK1), which is mediated primarily by the Kir2.1 protein. The use of renin-angiotensin-aldosterone system antagonists is associated with a reduced incidence of ventricular arrhythmias. Casein kinase 2 (CK2) binds and phosphorylates SP1, a transcription factor of KCNJ2 that encodes Kir2.1. Whether valsartan represses CK2 activation to ameliorate IK1 remodeling following MI remains unclear., Methods: Wistar rats suffering from MI received either valsartan or saline for 7 days. The protein levels of CK2 and Kir2.1 were each detected via a Western blot analysis. The mRNA levels of CK2 and Kir2.1 were each examined via quantitative real-time PCR., Results: CK2 expression was higher at the infarct border; and was accompanied by a depressed IK1/Kir2.1 protein level. Additionally, CK2 overexpression suppressed KCNJ2/Kir2.1 expression. By contrast, CK2 inhibition enhanced KCNJ2/Kir2.1 expression, establishing that CK2 regulates KCNJ2 expression. Among the rats suffering from MI, valsartan reduced CK2 expression and increased Kir2.1 expression compared with the rats that received saline treatment. In vitro, hypoxia increased CK2 expression and valsartan inhibited CK2 expression. The over-expression of CK2 in cells treated with valsartan abrogated its beneficial effect on KCNJ2/Kir2.1., Conclusions: AT1 receptor antagonist valsartan reduces CK2 activation, increases Kir2.1 expression and thereby ameliorates IK1 remodeling after MI in the rat model.

Neutrophils are phagocytic cells that play a critical role in innate immunity by destroying bacterial pathogens. Channels belonging to the inward rectifier potassium channel subfamily 2 (Kir2 channels) have been described in other phagocytes (monocytes/macrophages and eosinophils) and in hematopoietic precursors of phagocytes. Their physiological function in these cells remains unclear, but some evidence suggests a role in growth factor-dependent proliferation and development. Expression of functional Kir2 channels has not been definitively demonstrated in mammalian neutrophils. Here, we show by RT-PCR that neutrophils from mouse bone marrow and liver express mRNA for the Kir2 subunit Kir2.1 but not for other subunits (Kir2.2, Kir2.3, and Kir2.4). In electrophysiological experiments, resting (unstimulated) neutrophils from mouse bone marrow and liver exhibit a constitutively active, external K(+)-dependent, strong inwardly rectifying current that constitutes the dominant current. The reversal potential is dependent on the external K(+) concentration in a Nernstian fashion, as expected for a K(+)-selective current. The current is not altered by changes in external or internal pH, and it is blocked by Ba(2+), Cs(+), and the Kir2-selective inhibitor ML133. The single-channel conductance is in agreement with previously reported values for Kir2.1 channels. These properties are characteristic of homomeric Kir2.1 channels. Current density in short-term cultures of bone marrow neutrophils is decreased in the absence of growth factors that are important for neutrophil proliferation [granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF)]. These results demonstrate that mouse neutrophils express functional Kir2.1 channels and suggest that these channels may be important for neutrophil function, possibly in a growth factor-dependent manner., (Copyright © 2015 the American Physiological Society.)

Potassium inwardly rectifying channel, subfamily J, member 1 (KCNJ1), as an ATP-dependent potassium channel, plays an essential role in potassium balance. KCNJ1 variation is associated with multiple diseases, such as antenatal Bartter syndrome and diabetes. However, the role of KCNJ1 in clear cell renal cell carcinoma (ccRCC) is still unknown. Here, we studied the expression and function of KCNJ1 in ccRCC. The expression of KCNJ1 was evaluated in ccRCC tissues and cell lines by quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry analysis. The relationship between KCNJ1 expression and clinicopathological characteristics was analyzed. p3xFLAG-CMV-14 vector containing KCNJ1 was constructed and used for transfecting ccRCC cell lines 786-O and Caki-2. The effects of KCNJ1 on cell proliferation, invasion, and apoptosis were detected in ccRCC cell lines using cell proliferation assay, transwell assay, and flow cytometry, respectively. We found that KCNJ1 was low-expressed in ccRCC tissues samples and cell lines, and its expression level was significantly associated with tumor pathology grade (P = 0.002) and clinical stage (P = 0.023). Furthermore, the KCNJ1 expression was a prognostic factor of ccRCC patient's survival (P = 0.033). The re-expression of KCNJ1 in 786-O and Caki-2 significantly inhibited cancer cell growth and invasion and promoted cancer cell apoptosis. Moreover, knockdown of KCNJ1 in HK-2 cells promoted cell proliferation. Collectively, these data highlight that KCNJ1, low-expressed in ccRCC and associated with poor prognosis, plays an important role in ccRCC cell growth and metastasis.

Introduction: Thyroid morphogenesis is a complex process. Inwardly rectifying potassium (Kir) genes play a role in hormone release, cell excitability, pH and K(+) homeostasis in many tissues., Objectives: To investigate the thyroid developmental expression of three members, Kir4.1, Kir4.2 and Kir5.1, in mice. To postulate the K(+) channel role in thyroid hormone secretion., Material and Methods: Quantitative RT-PCR analysis of Kir4.1, Kir4.2 and Kir5.1 in mice of different stages (E13.5-E18.5)., Results: mRNA for Kir4.1, Kir4.2 and Kir5.1 were identified and increased with age in mice. Both Kir4.1 and Kir4.2 genes are better expressed after E16.5. Kir4.2 greatly increases from E13.5 to E16.5 (p ≤ 0.05)., Conclusion: Quantitative PCR shows that the mouse thyroid presents increased expression for Kir channels during development. The role of Kir in thyroid morphogenesis and differentiation might be understood in future studies. We speculate that thyroglobulin trafficking might be modulated by Kir4.1/5.1., (© 2015 S. Karger AG, Basel.)

Regulation of plasma K(+) levels in narrow ranges is vital to vertebrate animals. Since seawater (SW) teleosts are loaded with excess K(+), they constantly excrete K(+) from the gills. However, the K(+) regulatory mechanisms in freshwater (FW)-acclimated teleosts are still unclear. We aimed to identify the possible K(+) regulatory mechanisms in the gills and kidney, the two major osmoregulatory organs, of FW-acclimated Mozambique tilapia (Oreochromis mossambicus). As a potential molecular candidate for renal K(+) handling, a putative renal outer medullary K(+) channel (ROMK) was cloned from the tilapia kidney and tentatively named "ROMKb"; another ROMK previously cloned from the tilapia gills was thus renamed "ROMKa". The fish were acclimated to control FW or to high-K(+) (H-K) FW for 1 wk, and we assessed physiological responses of tilapia to H-K treatment. As a result, urinary K(+) levels were slightly higher in H-K fish, implying a role of the kidney in K(+) excretion. However, the mRNA expression levels of both ROMKa and ROMKb were very low in the kidney, while that of K(+)/Cl(-) cotransporter 1 (KCC1) was robust. In the gills, ROMKa mRNA was markedly upregulated in H-K fish. Immunofluorescence staining showed that branchial ROMKa was expressed at the apical membrane of type I and type III ionocytes, and the ROMKa immunosignals were more intense in H-K fish than in control fish. The present study suggests that branchial ROMKa takes a central role for K(+) regulation in FW conditions and that K(+) excretion via the gills is activated irrespective of environmental salinity., (Copyright © 2014 the American Physiological Society.)

Objective: Neovascularization and vaso-obliteration are vision-threatening events that develop by interactions between retinal vascular and glial cells. A high-salt diet is causal in cardiovascular and renal disease, which is linked to modulation of the renin-angiotensin-aldosterone system. However, it is not known whether dietary salt influences retinal vasculopathy and if the renin-angiotensin-aldosterone system is involved. We examined whether a low-salt (LS) diet influenced vascular and glial cell injury and the renin-angiotensin-aldosterone system in ischemic retinopathy., Approach and Results: Pregnant Sprague Dawley rats were fed LS (0.03% NaCl) or normal salt (0.3% NaCl) diets, and ischemic retinopathy was induced in the offspring. An LS diet reduced retinal neovascularization and vaso-obliteration, the mRNA and protein levels of the angiogenic factors, vascular endothelial growth factor, and erythropoietin. Microglia, which influence vascular remodeling in ischemic retinopathy, were reduced by LS as was tumor necrosis factor-α. Macroglial Müller cells maintain the integrity of the blood-retinal barrier, and in ischemic retinopathy, LS reduced their gliosis and also vascular leakage. In retina, LS reduced mineralocorticoid receptor, angiotensin type 1 receptor, and renin mRNA levels, whereas, as expected, plasma levels of aldosterone and renin were increased. The aldosterone/mineralocorticoid receptor-sensitive epithelial sodium channel alpha (ENaCα), which is expressed in Müller cells, was increased in ischemic retinopathy and reduced by LS. In cultured Müller cells, high salt increased ENaCα, which was prevented by mineralocorticoid receptor and angiotensin type 1 receptor blockade. Conversely, LS reduced ENaCα, angiotensin type 1 receptor, and mineralocorticoid receptor expression., Conclusions: An LS diet reduced retinal vasculopathy, by modulating glial cell function and the retinal renin-angiotensin-aldosterone system., (© 2014 American Heart Association, Inc.)

At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K(+) (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved μ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined μ {[d-Arg(2),Ly(4)]dermorphin-(1-4)-amide}- and δ ([d-Pen(2),5,P-Cl-Phe(4)]enkephalin)-opioid agonists (2 μM) for ∼7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers., (Copyright © 2014 the American Physiological Society.)

Background: Resveratrol is a natural compound that affects energy metabolism and is also known to possess an array of cardioprotective effects. However, its overall effects on energy metabolism and the underlying mechanism involved in cardioprotection require further investigation. Herein we hypothesize that ATP-sensitive potassium (K-ATP) channels as molecular sensors of cellular metabolism may mediate the cardioprotective effects of resveratrol., Methods: Kir6.2 knockout, Kir6.1 heterozygous and wild-type (WT) mice were subjected to ischemia/reperfusion injury and were injected with resveratrol (10 mg/kg, i.p). Myocardial infarct size, serum lactate dehydrogenase (LDH) and creatine kinase (CK) activities were determined. Neonatal cardiomyocytes were used in in vitro assays to investigate the underlying mechanism of resveratrol in cardioprotection., Results: Resveratrol treatment significantly reduced myocardial infarct size and serum LDH and CK activity and inhibited oxygen-glucose deprivation/reoxygenation - induced cardiomyocyte apoptosis in WT and Kir6.1 heterozygous mice, but Kir6.2 deficiency can abolish the cardioprotective effects of resveratrol in vivo and in vitro. We further found that resveratrol enhanced 5'-AMP-activated protein kinase (AMPK) phosphorylation and promoted the association of AMPK with Kir6.2. Suppression of AMPK attenuated and activation of AMPK mimicked the cardioprotective effects of resveratrol in cardiomyocytes. Notably, Kir6.2 knockout also reversed the cardioprotection of AMPK activator., Conclusions: Our study demonstrates that resveratrol exerts cardioprotective effects through AMPK -Kir6.2/K-ATP signal pathway and Kir6.2-containing K-ATP channel is required for cardioprotection of resveratrol.

K(+) channels in mammary secretory (MS) cells are believed to play a role in transcellular electrolyte transport and thus determining ionic composition of the aqueous phase of milk. However, direct evidence for specific K(+) channel activity in native MS cells is lacking at the single-cell level. Here, we show for the first time that an inwardly rectifying K(+) (Kir) channel is functionally expressed in fully differentiated MS cells that were freshly isolated from the mammary gland of lactating mice. Using the standard whole cell patch-clamp technique, we found that mouse MS cells consistently displayed a K(+) current, whose electrophysiological properties are similar to those previously reported for Kir2.x channels, particularly Kir2.1: 1) current-voltage relationship with strong inward rectification, 2) slope conductance approximately proportional to the square root of external K(+) concentration, 3) voltage- and time-dependent and high-affinity block by external Ba(2+), and 4) voltage-dependent inhibition by external Cs(+). Accordingly, RT-PCR analysis revealed the gene expression of Kir2.1, but not Kir2.2, Kir2.3, and Kir2.4, in lactating mouse mammary gland, and immunohistochemical staining showed Kir2.1 protein expression in the secretory cells. Cell-attached patch recordings from MS cells revealed that a 31-pS K(+) channel with strong inward rectification was likely active at the resting membrane potential. Collectively, the present work demonstrates that a functional Kir2.1-like channel is expressed in lactating mouse MS cells. We propose that the channel might be involved, at least in part, in secretion and/or preservation of ionic components of milk stored into the lumen of these cells.

The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8 mV/cm(2)) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.

Background/aims: The serum & glucocorticoid inducible kinase SGK3, an ubiquitously expressed serine/threonine kinase, regulates a variety of ion channels. It has previously been shown that SGK3 upregulates the outwardly rectifying K(+) channel KV11.1, which is expressed in cardiomyocytes. Cardiomyocytes further express the inward rectifier K(+) channel K(ir)2.1, which contributes to maintenance of resting cell membrane potential. Loss-of-function mutations of KCNJ2 encoding K(ir)2.1 result in Andersen-Tawil syndrome with periodic paralysis, cardiac arrhythmia and dysmorphic features. The present study explored whether SGK3 participates in the regulation of K(ir)2.1., Methods: cRNA encoding K(ir)2.1 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild type SGK3, constitutively active (S419D)SGK3 or inactive (K191N)SGK3. Kir2.1 activity was determined by two-electrode voltage-clamp and K(ir)2.1 protein abundance in the cell membrane by immunostaining and subsequent confocal imaging or by chemiluminescence., Results: Injection of 10 ng cRNA encoding wild type SGK3 and (S419D)SGK3, but not (K191N)SGK3 significantly enhanced K(ir)2.1-mediated currents. SGK inhibitor EMD638683 (50 µM) abrogated (S419D)SGK3-induced up-regulation of K(ir)2.1. Moreover, wild type SGK3 enhanced the channel protein abundance in the cell membrane. The decay of K(ir)2.1-mediated currents following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) was similar in oocytes coexpressing K(ir)2.1 and SGK3 as in oocytes expressing K(ir)2.1 alone, suggesting that SGK3 influences channel insertion into rather than channel retrieval from the cell membrane., Conclusions: SGK3 is a novel regulator of K(ir)2.1., (© 2014 S. Karger AG, Basel.)

Potassium channels modulate macrophage physiology. Blockade of voltage-dependent potassium channels (Kv) by specific antagonists decreases macrophage cytokine production and inhibits proliferation. In the presence of aspirin, acetylated cyclooxygenase-2 loses the activity required to synthesize PGs but maintains the oxygenase activity to produce 15R-HETE from arachidonate. This intermediate product is transformed via 5-LOX into epimeric lipoxins, termed 15-epi-lipoxins (15-epi-lipoxin A4 [e-LXA4]). Kv have been proposed as anti-inflammatory targets. Therefore, we studied the effects of e-LXA4 on signaling and on Kv and inward rectifier potassium channels (Kir) in mice bone marrow-derived macrophages (BMDM). Electrophysiological recordings were performed in these cells by the whole-cell patch-clamp technique. Treatment of BMDM with e-LXA4 inhibited LPS-dependent activation of NF-κB and IκB kinase β activity, protected against LPS activation-dependent apoptosis, and enhanced the accumulation of the Nrf-2 transcription factor. Moreover, treatment of LPS-stimulated BMDM with e-LXA4 resulted in a rapid decrease of Kv currents, compatible with attenuation of the inflammatory response. Long-term treatment of LPS-stimulated BMDM with e-LXA4 significantly reverted LPS effects on Kv and Kir currents. Under these conditions, e-LXA4 decreased the calcium influx versus that observed in LPS-stimulated BMDM. These effects were partially mediated via the lipoxin receptor (ALX), because they were significantly reverted by a selective ALX receptor antagonist. We provide evidence for a new mechanism by which e-LXA4 contributes to inflammation resolution, consisting of the reversion of LPS effects on Kv and Kir currents in macrophages.

Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been used to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel nonconducting, and, on brief light illumination, was released to permit outward K(+) current. Expression of this photoinducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expanded the genetic code of mammals to express PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. These principles could be generally expanded to other proteins expressed in the brain to enable optical regulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: To investigate the effects of testosterone on myocardial contractility, oxidative stress status and expression of sodium channel protein (Nav1.5) and inward rectifying K channels (Kir 2.x) in normal and orchidectomized (ORX) rats., Methods: One hundred four rats were randomly assigned into four groups (n = 26, each) as follows: (i) untreated controls, (ii) testosterone treated, (iii) orchidectomized rats and (iv) orchidectomized, testosterone-treated rats. Treatments with the vehicle or testosterone were carried out for 12 weeks, three times per week. At the end of treatment, surface ECG, isolated heart, tissue oxidative stress and lipid peroxidation experiments were carried out on the cardiac tissues. Also, immunohistochemical examination for Nav1.5 and PCR detection of mRNA of Kir2.1, Kir2.2 and Kir2.4 subunits of K channels were carried out., Results: Orchidectomy impaired cardiac contractile function parameters left ventricular developed pressure (LVDP) and the peaks of the positive and negative pressure derivatives (dP/dtmax and -dP/dtmax respectively), increased heart rate and prolonged QT and QTc intervals, elevated pro-oxidant state in rat's hearts and decreased the expression of Kir 2.1 but not Kir2.2, Kir 2.4 and Nav1.5 channels. Exogenous testosterone administration to orchidectomized rats restored heart contractility and shortened QT and QTc intervals to their normal values, ameliorated the generated pro-oxidant state and improved the expression of Nav1.5 and Kir2.1, but not Kir2.2 or Kir2.4 channels., Conclusion: Testosterone improved cardiac contractility and shortened QT and QTc intervals in ORX rats. An effect that might be dependent of reduction in oxidative stress and enhancement of Kir2.1 channels but independent of Nav1.5 channel protein., (© 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Objective: Activating mutations in the KCNJ11 gene, encoding the Kir6.2 subunit of the KATP channel, result in permanent neonatal diabetes mellitus. They also may cause neurologic symptoms such as mental retardation and motor problems (iDEND syndrome) and epilepsy (DEND syndrome). Sulphonylurea (SU) treatment is reported to alleviate both the neurologic symptoms and diabetes in such cases. The study aimed to establish the magnitude and functional basis of the effect of SUs on the neurologic phenotype in children with iDEND using neuroimaging before and after insulin replacement with glibenclamide., Research Design and Methods: To localize and quantify the effect of glibenclamide administration, we performed single-photon emission computed tomography in seven patients with different mutations in KCNJ11. In five patients, measurements before and after initiation of SU treatment were performed. RESULTS Significant changes in single-photon emission computed tomography signal intensity after transfer to SU therapy were restricted to the cerebellum, consistent with previous data showing high Kir6.2 expression in this brain region. Cerebellar perfusion improved for both left (P = 0.006) and right (P = 0.01) hemispheres, with the mean improvement being 26.7 ± 7.1% (n = 5). No patients showed deterioration of cerebellar perfusion on SU therapy. Electrophysiological studies revealed a good correlation between the magnitude of KATP channel dysfunction and the clinical phenotype; mutant channels with the greatest reduction in adenosine 5'-triphosphate inhibition were associated with the most severe neurologic symptoms., Conclusions: We conclude it is likely that at least some of the beneficial effects of SU treatment on neurodevelopment in iDEND patients result from improved cerebellar perfusion.

Objective: Inwardly rectifying K(+) (Kir) channel, Kir2.3, has been found down-regulated in the hippocampus of chronic temporal lobe epileptic (cTLE) patients which may underline the mechanism of epilepsy. Tenidap is an agonist of Kir2.3. However, the effect of tenidap on cTLE remains obscure. The aim of the present study was to observe the relationship between Kir2.3 and the pathogenesis of cTLE, and to explore the potential effect of Kir agonists as anti-epileptic drugs., Methods: The pilocarpine temporal lobe epilepsy (TLE) rat model was established and status epilepticus (SE) was induced in rats. The rats were divided into four groups based on time points (0 hours, 6 hours, 72 hours, and two weeks) after SE termination. The dynamic changes in Kir2.3 mRNA and protein expression in the hippocampus were detected at each time point by reverse transcription polymerase chain reaction (RT-PCR) and western blotting to determine an appropriate time for further intervention and observation. Then, tenidap was administered and its effect on the expression of Kir2.3 mRNA/protein and the electroencephalogram (EEG) wave was documented at predetermined time points., Results: The pattern of Kir2.3 mRNA and protein expression was bimodal, as it increased immediately after SE and declined at two weeks, when compared with the control group. Down-regulation of the Kir channels may lead to an impaired clearance of K(+) ions from the extracellular space and result in a stronger neuronal depolarisation. Two weeks after SE may be a reversal point. Spontaneous recurrent seizures (SRS) appeared at the two-week point, indicating that chronic epilepsy had occurred. Tenidap up-regulated expressions of both Kir2.3 channel mRNA and protein, which may help to maintain the resting membrane potential (RMP) and hyperpolarisation of the cells., Conclusions: Our results suggest that the down-regulation of the Kir2.3 channel expression might contribute to the pathogenesis of TLE, which may be ameliorated by the administration of tenidap.

Stomata open in response to light and close after exposure to abscisic acid (ABA). They regulate gas exchange between plants and the atmosphere, enabling plants to adapt to changing environmental conditions. ABA binding to receptors initiates a signaling cascade that involves protein phosphorylation. We show that ABA induced the phosphorylation of three basic helix-loop-helix (bHLH) transcription factors, called AKSs (ABA-responsive kinase substrates; AKS1, AKS2, and AKS3), in Arabidopsis guard cells. In their unphosphorylated state, AKSs facilitated stomatal opening through the transcription of genes encoding inwardly rectifying K⁺ channels. aks1aks2-1 double mutant plants showed decreases in light-induced stomatal opening, K⁺ accumulation in response to light, activity of inwardly rectifying K⁺ channels, and transcription of genes encoding major inwardly rectifying K⁺ channels without affecting ABA-mediated stomatal closure. Overexpression of potassium channel in Arabidopsis thaliana 1 (KAT1), which encodes a major inwardly rectifying K⁺ channel in guard cells, rescued the phenotype of aks1aks2-1 plants. AKS1 bound directly to the promoter of KAT1, an interaction that was attenuated after ABA-induced phosphorylation. The ABA agonist pyrabactin induced phosphorylation of AKSs. Our results demonstrate that the AKS family of bHLH transcription factors facilitates stomatal opening through the transcription of genes encoding inwardly rectifying K⁺ channels and that ABA suppresses the activity of these channels by triggering the phosphorylation of AKS family transcription factors.

Background: Neuromodulation is used to restore neural function in disorders that stem from an imbalance in the activity of specific neural networks when they prove refractory to pharmacological therapy. The Kir2.1 gene contributes to stabilizing the resting potential below the threshold of activation of voltage-gated sodium channels and action potentials. Therefore, the delivery of the Kir2.1 gene to neuronal cells could reduce the probability of action potential generation, inhibiting excessive neural activity., Objective: To address the hypothesis that overexpression of the inwardly rectifying potassium channel 2.1 (Kir2.1) gene could inhibit motor neuron activity and therefore be therapeutically used in gene-based neuromodulation., Methods: To induce expression of Kir2.1, the inducible RheoSwitch promoter was used and controlled by ligand. In vivo gene expression was accomplished by an adenoviral vector to deliver unilaterally into the lumbar spinal cord of rats., Results: Behavioral assays demonstrated that neuromuscular inhibition was exclusive to rats that received the ligand. Histological analysis also showed evidence of some motor neuron loss in these animals. Behavioral effects of Kir2.1 expression were completely reversible, arguing that the behavioral effect did not result from motor neuron death., Conclusion: Delivery of the gene for Kir2.1 inhibits neurons by resisting depolarization to the action potential threshold. Regulated neuronal expression of Kir2.1 may provide an elegant means for neuromodulation in a selected neuronal population.

Metabolic modulation of neuronal excitability is becoming increasingly important as an antiepileptic therapy. It was reported that the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) and the activation of the ATP-sensitive potassium ion channel (K(ATP) channel) had an antiepileptic effect in models of epilepsy. To explore whether 2-DG exerts an antiepileptic effect through upregulation of the K(ATP) channel subunits Kir6.1 and Kir6.2, the expression of these subunits in hippocampus of five groups of mice with pilocarpine-induced status epilepticus (SE) was evaluated. A seizure group with pilocarpine-kindling convulsions (EP) was compared to similar groups treated with high, medium, and low 2-DG concentrations (100-500 mg/kg) and a normal control group (Con). Kir6.1 and Kir6.2 mRNAs and proteins were analyzed at 4 h, 1 days (acute period), 7 days (latent period), 30, and 60 days (chronic period) following SE. In the seizure group (compared to the Con group), hippocampal expression of Kir6.1 and Kir6.2 increased dramatically at 1, 7, and 30 days, and was further increased after treatment with medium and high dose 2-DG (all P < 0.05). Our results suggest that 2-DG may exert an antiepileptic effect through up-regulation of mRNAs and protein levels of Kir6.1 and Kir6.2, which may therefore be used as molecular targets in the treatment of epilepsy with 2-DG.

Oligodendrocyte precursor cells (OPCs) are the major source of myelinating oligodendrocytes during development. These progenitors are highly abundant at birth and persist in the adult where they are distributed throughout the brain. The large abundance of OPCs after completion of myelination challenges their unique role as progenitors in the healthy adult brain. Here we show that adult OPCs of the barrel cortex sense fine extracellular K(+) increases generated by neuronal activity, a property commonly assigned to differentiated astrocytes rather than to progenitors. Biophysical, pharmacological, and single-cell RT-PCR analyses demonstrate that this ability of OPCs establishes itself progressively through the postnatal upregulation of Kir4.1 K(+) channels. In animals with advanced cortical myelination, extracellular stimulation of layer V axons induces slow K(+) currents in OPCs, which amplitude correlates with presynaptic action potential rate. Moreover, using paired recordings, we demonstrate that the discharge of a single neuron can be detected by nearby adult OPCs, indicating that these cells are strategically located to detect local changes in extracellular K(+) concentration during physiological neuronal activity. These results identify a novel unitary neuron-OPC connection, which transmission does not rely on neurotransmitter release and appears late in development. Beyond their abundance in the mature brain, the postnatal emergence of a physiological response of OPCs to neuronal network activity supports the view that in the adult these cells are not progenitors only.

Glial cells, besides participating as passive supporting matrix, are also proposed to be involved in the optimization of the interstitial space for synaptic transmission by tight control of ionic and water homeostasis. In adult mouse brain, inwardly rectifying K+ (Kir4.1) and aquaporin-4 (AQP4) channels localize to astroglial endfeets in contact with brain microvessels and glutamate synapses, optimizing clearance of extracellular K(+) and water from the synaptic layers. However, it is still unclear whether there is an age-dependent difference in the expressions of Kir4.1 and AQP4 channels specifically during postnatal development and aging when various marked changes occur in brain and if these changes region specific. RT-PCR and immunoblotting was conducted to compare the relative expression of Kir4.1 and AQP4 mRNA and protein in the early and mature postnatal (0-, 15-, 45-day), adult (20-week), and old age (70-week) mice cerebral and cerebellar cortices. Expressions of Kir4.1 and AQP4 mRNA and protein are very low at 0-day. A pronounced and continuous increase was observed by mature postnatal ages (15-, 45-days). However, in the 70-week-old mice, expressions are significantly up-regulated as compared to 20-week-old mice. Both genes follow the same age-related pattern in both cerebral and cerebellar cortices. The time course and expression pattern suggests that Kir4.1 and AQP4 channels may play an important role in brain K(+) and water homeostasis in early postnatal weeks after birth and during aging.

Analysis of ion channel mutants is a widely used approach for dissecting ion channel function and for characterizing the mechanisms of action of channel-directed modulators. Expression of functional potassium channels in potassium-uptake-deficient yeast together with genetic selection approaches offers an unbiased, high-throughput, activity-based readout that can rapidly identify large numbers of active ion channel mutants. Because of the assumption-free nature of the method, detailed biophysical analysis of the functional mutants from such selections can provide new and unexpected insights into both ion channel gating and ion channel modulator mechanisms. Here, we present detailed protocols for generation and identification of functional mutations in potassium channels using yeast selections in the potassium-uptake-deficient strain SGY1528. This approach is applicable for the analysis of structure-function relationships of potassium channels from a wide range of sources including viruses, bacteria, plants, and mammals and can be used as a facile way to probe the interactions between ion channels and small-molecule modulators.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. Dysfunction and death of motor neurons are closely related to the modified astrocytic environment. Astrocytic endfeet, lining the blood-brain barrier (BBB), are enriched in two proteins, aquaporin-4 (AQP4) and inwardly rectifying potassium channel (Kir) 4.1. Both channels are important for the maintainance of a functional BBB astrocytic lining. In this study, expression levels of AQP4 and Kir4.1 were for the first time examined in the brainstem and cortex, along with the functional properties of Kir channels in cultured cortical astrocytes of the SOD1(G93A) rat model of ALS. Western blot analysis showed increased expression of AQP4 and decreased expression of Kir4.1 in the brainstem and cortex of the ALS rat. In addition, higher immunoreactivity of AQP4 and reduced immunolabeling of Kir4.1 in facial and trigeminal nuclei as well as in the motor cortex were also observed. Particularly, the observed changes in the expression of both channels were retained in cultured astrocytes. Furthermore, whole-cell patch-clamp recordings from cultured ALS cortical astrocytes showed a significantly lower Kir current density. Importantly, the potassium uptake current in ALS astrocytes was significantly reduced at all extracellular potassium concentrations. Consequently, the Kir-specific Cs(+)- and Ba(2+)-sensitive currents were also decreased. The changes in the studied channels, notably at the upper CNS level, could underline the hampered ability of astrocytes to maintain water and potassium homeostasis, thus affecting the BBB, disturbing the neuronal microenvironment, and causing motoneuronal dysfunction and death., (Copyright © 2012 Wiley Periodicals, Inc.)

ATP-sensitive K(+) (K(ATP)) channel subunits SUR2A and SUR2B in the rat brain were investigated by RT-PCR assay, western blot analysis, in situ hybridization histochemistry, and immunohistochemical staining. The results show that the mRNA and protein of SUR2A and SUR2B are expressed in whole rat brain extracts and selected regions. SUR2 mRNA is widely expressed in many neurons and glial cells as revealed by in situ hybridization histochemistry. Immunohistochemical staining shows SUR2A to be widely expressed in neurons of the brain, especially in the large pyramidal neurons and their main dendrites in the neocortex and in the Purkinje cells of the cerebellar cortex. In contrast to SUR2A, SUR2B is potently expressed in small cells in the corpus callosum and cerebellar white matter, but is also weakly expressed in some neurons. Double immunostaining shows SUR2B to be localized in astrocytes and oligodendrocytes, while SUR2A is only localized in oligodendrocytes. These results suggest that SUR2A might be mainly a regulatory subunit of the K(ATP) channel in most neurons and part of oligodendrocytes, while SUR2B might be mainly a regulatory subunit of the K(ATP) channel in astrocytes, oligodendrocytes, and some neurons., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

The sulfonylurea receptor 1 (Sur1)-regulated NC(Ca-ATP) channel is a nonselective cation channel that is regulated by intracellular calcium and adenosine triphosphate. The channel is not constitutively expressed, but is transcriptionally upregulated de novo in all cells of the neurovascular unit, in many forms of central nervous system (CNS) injury, including cerebral ischemia, traumatic brain injury (TBI), spinal cord injury (SCI), and subarachnoid hemorrhage (SAH). The channel is linked to microvascular dysfunction that manifests as edema formation and delayed secondary hemorrhage. Also implicated in oncotic cell swelling and oncotic (necrotic) cell death, the channel is a major molecular mechanism of 'accidental necrotic cell death' in the CNS. In animal models of SCI, pharmacological inhibition of Sur1 by glibenclamide, as well as gene suppression of Abcc8, prevents delayed capillary fragmentation and tissue necrosis. In models of stroke and TBI, glibenclamide ameliorates edema, secondary hemorrhage, and tissue damage. In a model of SAH, glibenclamide attenuates the inflammatory response due to extravasated blood. Clinical trials of an intravenous formulation of glibenclamide in TBI and stroke underscore the importance of recent advances in understanding the role of the Sur1-regulated NC(Ca-ATP) channel in acute ischemic, traumatic, and inflammatory injury to the CNS.

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.

Fetal nicotine exposure causes impaired adrenal catecholamine secretion and increased neonatal mortality during acute hypoxic challenges. Both effects are attributable to upregulation of ATP-sensitive K(+) channels (K(ATP) channels) and can be rescued by pretreatment with the blocker, glibenclamide. Although use of in vitro models of primary and immortalized, fetal-derived rat adrenomedullary chromaffin cells (i.e., MAH cells) demonstrated the involvement of α7 nicotinic ACh receptor (nAChR) stimulation and the transcription factor, HIF-2α, the latter's role was unclear. Using Western blots, we show that chronic nicotine causes a progressive, time-dependent induction of HIF-2α in MAH cells that parallels the upregulation of K(ATP) channel subunit, Kir6.2. Moreover, a common HIF target, VEGF mRNA, was also upregulated after chronic nicotine. All the above effects were prevented during co-incubation with α-bungarotoxin (100 nM), a specific α7 nAChR blocker, and were absent in HIF-2α-deficient MAH cells. Chromatin immunoprecipitation (ChIP) assays demonstrated binding of HIF-2α to a putative hypoxia response element in Kir6.2 gene promoter. Specificity of this signaling pathway was validated in adrenal glands from pups born to dams exposed to nicotine throughout gestation; the upregulation of both HIF-2α and Kir6.2 was confined to medullary, but not cortical, tissue. This study has uncovered a signaling pathway whereby a nonhypoxic stimulus (nicotine) promotes HIF-2α-mediated transcriptional upregulation of a novel target, Kir6.2 subunit. The data suggest that the HIF pathway may be involved in K(ATP) channel-mediated neuroprotection during brain ischemia, and in the effects of chronic nicotine on ubiquitous brain α7 nAChR.