Upregulation Of Kir2 Research Articles

The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity

The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomics screen for polysome-associated proteins in astrocytes and focused on ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially binds to a number of mRNAs, including Kcnj10, encoding the inward-rectifying potassium (K+) channel Kir4.1. By developing an astrocyte-specific, conditional RACK1 knockout mouse model, we show that RACK1 represses production of Kir4.1 in hippocampal astrocytes and PAPs. Upregulation of Kir4.1 in the absence of RACK1 increases astrocytic Kir4.1-mediated K+ currents and volume. It also modifies neuronal activity attenuating burst frequency and duration. Reporter-based assays reveal that RACK1 controls Kcnj10 translation through the transcript's 5' untranslated region. Hence, translational regulation by RACK1 in astrocytes represses Kir4.1 expression and influences neuronal activity.

Hypoxia Induces Apoptosis of Microglia BV2 by Upregulating Kir2.1 to Activate Mitochondrial-Related Apoptotic Pathways

Aim To explore the role of Kir2.1 in hypoxia-induced microglial apoptosis. Methods BV2 microglial cell lines were cultured and treated with ML133 hydrochloride, a Kir2.1 channel blocker, for 23 h and with 500 μmol/L of CoCl2 for 8 h. Cells were divided into the control, CoCl2 (hypoxia-induced model), and CoCl2+ML133 (hypoxia-induced model established after ML133 pretreatment) groups. Cell activity was assessed using the CCK-8 technique. The membrane potential and Kir2.1 current of BV2 were evaluated with the whole-cell patch-clamp technique. The protein levels and mRNA levels of Kir2.1, apoptotic proteins Bax and caspase-3, and antiapoptotic protein Bcl-2 in BV2 cells were evaluated via immunofluorescence, Western blot analysis, and real-time quantitative reverse transcription. The apoptosis rate of BV2 cells was detected via flow cytometry. Results CCK-8 analysis showed that the cell activity of each group increased initially and then decreased. The 2 h intervention group had the highest cell activity, and that of the 8 h group was >90%. Hence, there was a significant difference in the results (P < 0.05). Western blot analysis revealed that the expression of cleaved caspase-3 significantly increased in the 8 h group compared with the 0 h group. Compared with the control group, the expression of Kir2.1 and mRNA in the CoCl2 group increased. Thus, hypoxia could upregulate the expression of Kir2.1. The whole-cell patch-clamp results showed that the Kir2.1 channel current amplitude of the CoCl2 group increased compared with that of the control group. Therefore, hypoxia could enhance Kir2.1 function. The apoptosis rate of the CoCl2 group was significantly higher than that of the control group. Further, the ML133 group had a significantly lower apoptosis rate than the CoCl2 group. The expression of apoptotic proteins Bax and cleaved caspase-3 increased in the CoCl2 group, and that of the antiapoptotic protein Bcl-2 decreased. The expression of apoptotic proteins Bax and cleaved caspase-3 reduced in the CoCl2+ML133 group, whereas that of the antiapoptotic protein Bcl-2 increased. Conclusion Hypoxia can induce microglia BV2 apoptosis accompanied by the upregulation of Kir2.1 and mRNA expression levels and an increase in the Kir2.1 current. Moreover, ML133 can inhibit hypoxia-induced BV2 cell apoptosis. Hence, Kir2.1 may be involved in the process of hypoxia-induced BV2 cell apoptosis.

Slight up‐regulation of Kir2.1 channel promotes endothelial progenitor cells to transdifferentiate into a pericyte phenotype by Akt/mTOR/Snail pathway

It was shown that endothelial progenitor cells (EPCs) have bidirectional differentiation potential and thus perform different biological functions. The purpose of this study was to investigate the effects of slight up‐regulation of the Kir2.1 channel on EPC transdifferentiation and the potential mechanism on cell function and transformed cell type. First, we found that the slight up‐regulation of Kir2.1 expression promoted the expression of the stem cell stemness factors ZFX and NS and inhibited the expression of senescence‐associated β‐galactosidase. Further studies showed the slightly increased expression of Kir2.1 could also improve the expression of pericyte molecular markers NG2, PDGFRβ and Desmin. Moreover, adenovirus‐mediated Kir2.1 overexpression had an enhanced contractile response to norepinephrine of EPCs. These results suggest that the up‐regulated expression of the Kir2.1 channel promotes EPC transdifferentiation into a pericyte phenotype. Furthermore, the mechanism of EPC transdifferentiation to mesenchymal cells (pericytes) was found to be closely related to the channel functional activity of Kir2.1 and revealed that this channel could promote EPC EndoMT by activating the Akt/mTOR/Snail signalling pathway. Overall, this study suggested that in the early stage of inflammatory response, regulating the Kir2.1 channel expression affects the biological function of EPCs, thereby determining the maturation and stability of neovascularization.

Anti-VEGF therapy prevents M\xfcller intracellular edema by decreasing VEGF-A in diabetic retinopathy

BackgroundAlthough vascular endothelial growth factor A (VEGF-A) is known to play a key role in causing retinal edema, whether and how VEGF-A induces intracellular edema in the retina still remains unclear.MethodsSprague-Dawley rats were rendered diabetic with intraperitoneal injection of streptozotocin. Intravitreal injection of ranibizumab was performed 8 weeks after diabetes onset. rMC-1 cells (rat Müller cell line) were treated with glyoxal for 24 h with or without ranibizumab. The expression levels of inwardly rectifying K+ channel 4.1 (Kir4.1), aquaporin 4 (AQP4), Dystrophin 71 (Dp71), VEGF-A, glutamine synthetase (GS) and sodium-potassium-ATPase (Na+-K+-ATPase) were examined using Western blot. VEGF-A in the supernatant of the cell culture was detected with ELISA. The intracellular potassium and sodium levels were detected with specific indicators.ResultsCompared with normal control, protein expressions of Kir4.1 and AQP4 were down-regulated significantly in diabetic rat retinas, which were prevented by ranibizumab. The above changes were recapitulated in vitro. Similarly, the intracellular potassium level in glyoxal-treated rMC-1 cells was increased, while the intracellular sodium level and Na+-K+-ATPase protein level remained unchanged, compared with control. However, ranibizumab treatment decreased intracellular sodium, but not potassium.ConclusionRanibizumab protected Müller cells from diabetic intracellular edema through the up-regulation of Kir4.1 and AQP4 by directly binding VEGF-A. It also caused a reduction in intracellular osmotic pressure.

Textured nanofibrils drive microglial phenotype

Microglia are highly plastic cells that change their properties in response to their microenvironment. By using immunofluorescence, live-cell imaging, electrophysiological recordings and RNA sequencing, we investigated the regulation of modified bacterial cellulose (mBC) nanofibril substrates on microglial properties. We demonstrate that mBC substrates induce ramified microglia with constantly extending and retracting processes, reminiscent of what is observed in vivo. Patch-clamp recordings show that microglia acquire a more negative resting membrane potential and have increased inward rectifier K+ currents, caused by an upregulation of Kir2.1 channels. Transcriptome analysis shows upregulation of genes involved in the immune response and downregulation of genes linked to cell adhesion and cell motion. Furthermore, Arp2/3 complex activation and integrin-mediated signaling modulate microglial morphology and motility. Our studies demonstrate that mBC nanofibril substrates modulate microglial phenotype, paving the way for a microglia-material interface that may be very valuable for anti-neuroinflammatory drug screening.

Magnesium Deficiency Causes Transcriptional Downregulation of Kir2.1 and Kv4.2 Channels in Cardiomyocytes Resulting in QT Interval Prolongation.

Mechanisms for QT interval prolongation and cardiac arrhythmogenesis in hypomagnesemia are poorly understood. This study investigated the potential molecular mechanism for QT prolongation caused by magnesium (Mg) deficiency in rats by using the patch clamp technique and molecular biology. Male Wistar rats were fed an Mg-free diet or a normal diet for up to 12 weeks. There was QT prolongation in the ECG of Mg-deficient rats, and cardiomyocytes from these rats showed prolongation of action potential duration. Electrophysiological studies showed that inward-rectifying K+current (IK1) and transient outward K+current (Ito) were decreased in Mg-deficient cardiomyocytes, and these findings were consistent with the downregulation of mRNA, as well as protein levels of Kir2.1 and Kv4.2. In Mg-deficient cardiomyocytes, transcription factors, GATA4 and NFAT, were upregulated, whereas CREB was downregulated. In contrast to Mg deficiency, cellular Mg2+overload in cultured cardiomyocytes resulted in the upregulation of Kir2.1 and Kv4.2, which was accompanied by the downregulation of GATA4 and NFATc4, and the upregulation of CREB. Activation of NFAT and inhibition of CREB reduced Kv4.2-Ito, whereas Kir2.1-IK1was reduced by CREB inhibition but not by NFTA activation. Intracellular Mg deficiency downregulates IK1and Itoin cardiomyocytes, and this is mediated by the transcription factors, NFAT and CREB. These results provide a novel mechanism for the long-term QT interval prolongation in hypomagnesemia.

Kir4.1/Kir5.1 Activity Is Essential for Dietary Sodium Intake-Induced Modulation of Na-Cl Cotransporter.

Dietary sodium intake regulates the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Whether the basolateral, inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1/Kir5.1) in the DCT is essential for mediating the effect of dietary sodium intake on NCC activity is unknown. We used electrophysiology, renal clearance techniques, and immunoblotting to examine effects of Kir4.1/Kir5.1 in the DCT and NCC in wild-type and kidney-specific Kir4.1 knockout mice. Low sodium intake stimulated basolateral Kir4.1/Kir5.1 activity, increased basolateral K+ conductance, and hyperpolarized the membrane. Conversely, high sodium intake inhibited the potassium channel, decreased basolateral K+ currents, and depolarized the membrane. Low sodium intake increased total and phosphorylated NCC expression and augmented hydrochlorothiazide-induced natriuresis; high sodium intake had opposite effects. Thus, elevated NCC activity induced by low sodium intake was associated with upregulation of Kir4.1/Kir5.1 activity in the DCT, whereas inhibition of NCC activity by high sodium intake was associated with diminished Kir4.1/Kir5.1 activity. In contrast, dietary sodium intake did not affect NCC activity in knockout mice. Further, Kir4.1 deletion not only abolished basolateral K+ conductance and depolarized the DCT membrane, but also abrogated the stimulating effects induced by low sodium intake on basolateral K+ conductance and hyperpolarization. Finally, dietary sodium intake did not alter urinary potassium excretion rate in hypokalemic knockout and wild-type mice. Stimulation of Kir4.1/Kir5.1 by low intake of dietary sodium is essential for NCC upregulation, and inhibition of Kir4.1/Kir5.1 induced by high sodium intake is a key step for downregulation of NCC.

Antiepileptic Drugs Elevate Astrocytic Kir4.1 Expression in the Rat Limbic Region.

Inwardly rectifying potassium (Kir) channel subunits Kir4.1 are specifically expressed in astrocytes and regulate neuronal excitability by mediating spatial potassium buffering. In addition, it is now known that astrocytic Kir4.1 channels are closely involved in the pathogenesis of epilepsy. Here, to explore the role of Kir4.1 channels in the treatment of epilepsy, we evaluated the effects of the antiepileptic drugs, valproate, phenytoin, phenobarbital and ethosuximide, on Kir4.1 expression in astrocytes using immunohistochemical techniques. Repeated treatment of rats with valproate (30–300 mg/kg, i.p., for 1–10 days) significantly elevated the Kir4.1 expression levels in the cerebral cortex, amygdala and hippocampus. Up-regulation of Kir4.1 expression by valproate occurred in a dose- and treatment period-related manner, and did not accompany an increase in the number of astrocytes probed by glial fibrillary acidic protein (GFAP). In addition, repeated treatment with phenytoin (30 mg/kg, i.p., for 10 days) or phenobarbital (30 mg/kg, i.p., for 10 days) also elevated Kir4.1 expression region-specifically in the amygdala. However, ethosuximide (100 mg/kg, i.p., for 10 days), which can alleviate absence but not convulsive seizures, showed no effects on the astrocytic Kir4.1 expression. The present results demonstrated for the first time that the antiepileptic drugs effective for convulsive seizures (valproate, phenytoin, and phenobarbital) commonly elevate the astrocytic Kir4.1 channel expression in the limbic regions, which may be related to their antiepileptic actions.

Kir4.1 activity is essential for dietary Na+ intake induced modulation of Na‐Cl cotransporter (NCC)

Dietary Na+ intake has been shown to modulate the thiazide‐sensitive NCC in the distal convoluted tubule (DCT), such that a high Na+ intake (HNa) inhibits whereas a low Na+ diet (LNa) stimulates NCC activity. However, the mechanism by which dietary Na+ intake on NCC is not clear. The aim of the present study is to test the hypothesis Kir4.1 is essential for mediating the effect of dietary Na+ intake on NCC. The patch‐clamp experiments demonstrated that the basolateral 40 pS K+ channel (a Kir4.1/Kir5.1 heterotetramer) activity in the DCT was upregulated in the mice on a LNa diet for 7 days but it was down‐regulated in the mice on HNa for 7 days. The whole‐cell recording further demonstrated that HNa decreased basolateral K+ conductance of the DCT and depolarized the membrane. Conversely, LNa intake augmented the basolateral K+ currents and hyperpolarized the DCT membrane. Western blot and renal clearance studies demonstrated that LNa increased the expression of total NCC (tNCC) and phosphorylated NCC (pNCC) and enhanced hydrochlorothiazide (HCTZ)‐induced natriuresis while HNa intake had opposite effects on the expression of tNCC and pNCC and reduced HCTZ‐induced natriuresis. Thus, an upregulation of Kir4.1 is correlated with a high NCC activity in the mice on LNa while a down‐regulation of Kir4.1 is related with low NCC activity in the mice on HNa. To explore the role of Kir4.1 in mediating the effect of dietary Na+ intake on NCC, we repeated the above experiments in kidney‐specific conditional Kir4.1 knockout mice (KS‐Kir4.1 KO). The deletion of Kir4.1 completely abolished the basolateral K+ conductance and depolarized the DCT membrane. Moreover, the effect of dietary Na+ intake on the basolateral K+ conductance and membrane potential was also absent in KS‐Kir4.1 KO mice. Also, the deletion of Kir4.1 decreased the expression of both tNCC and pNCC and abolished HCTZ‐induced natriuresis. Western blot and renal clearance experiments showed that the effect of dietary Na+ intake on NCC expression and activity was absent in KS‐Kir4.1 KO mice. We conclude that Kir4.1 plays an essential role in mediating the effect of dietary Na+ intake on NCC activity such that LNa‐induced stimulation of Kir4.1 in the DCT is essential for the upregulation of NCC while HNa‐induced inhibition of Kir4.1 is required for the down‐regulation of NCC.Support or Funding InformationNIH DK54983This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Kir2.1 in human monocyte‐derived foam cell maturation

The role of K(+) channels in macrophage immunomodulation has been well-established. However, it remains unclear whether K(+) channels are involved in the lipid uptake of macrophages. The expression and function of the inward rectifier potassium channel (Kir2.1, KCNJ2) in Human acute monocytic leukemia cell line (THP-1) cells and human monocytes derived macrophages (HMDMs) were investigated using RT-PCR and western blotting, and patch clamp technique. The expression of scavenger receptors in THP-1-derived macrophages was detected using western blotting. Expressions of Kir2.1 mRNA and protein in HMDMs were significantly decreased by 60% (P < 0.05) and 90% (P < 0.001) on macrophage maturation, but overexpressed by approximately 1.3 (P > 0.05) and 3.8 times (P = 0.001) after foam cell formation respectively. Concurrently, the Kir2.1 peak current density in HMDMs, mature macrophages and foam cells, measured at -150 mV, were -22.61 ± 2.1 pA/pF, -7.88 ± 0.60 pA/pF and -13.39 ± 0.80 pA/pF respectively (P < 0.05). In association with an up-regulation of Kir2.1 in foam cells, the SR-A protein level was significantly increased by over 1.5 times compared with macrophages (P < 0.05). THP-1 cells contained much less lipids upon Kir2.1 knockdown and cholesterol ester/total cholesterol ratio was 29.46 ± 2.01% (P < 0.05), and the SR-BI protein level was increased by over 6.2 times, compared to that of macrophages (P < 0.001). Kir2.1 may participate in macrophage maturation and differentiation, and play a key role in lipid uptake and foam cell formation through modulating the expression of scavenger receptors.

Müller glia activation by VEGF-antagonizing drugs: An in vitro study on rat primary retinal cultures

The effects of the anti-Vascular Endothelial Growth Factor (VEGF) drugs ranibizumab and aflibercept were studied in Müller glia in primary mixed cultures from rat neonatal retina. Treatment with both agents induced activation of Müller glia, demonstrated by increased levels of Glial Fibrillary Acidic Protein. In addition, phosphorylated Extracellular-Regulated Kinase 1/2 (ERK 1/2) showed enhanced immunoreactivity in activated Müller glia. Treatment with aflibercept induced an increase in K+ channel (Kir) 4.1 levels and both drugs upregulated Aquaporin 4 (AQP4) in activated Müller glia. The results show that VEGF-antagonizing drugs influence the homeostasis of Müller cells in primary retinal cultures, inducing an activated phenotype. Upregulation of Kir4.1 and AQP4 suggests that Müller glia activation following anti-VEGF drugs may not depict a detrimental gliotic reaction. Indeed, it could represent one of the mechanisms able to contribute to the therapeutic effects of these drugs, particularly in the presence of macular edema.

Kir2.1 Channels Compensate for the Loss of KATP Channels in SUR1 Null Islets

Pancreatic islets of SUR1-/- mice exhibit electrical oscillations even though they lack KATP channels. However, while we confirmed that electrical oscillations persisted in freshly isolated SUR1-/- islets, after overnight culture in media containing 11.1 mM glucose, SUR1-/- islets exhibited continuous spiking in place of oscillations, as has been observed in human congenital hyperinsulinemia (CHI). Microarray analysis suggested upregulation of kcnj2, the gene encoding the inward rectifier Kir2.1, in SUR1-/- islets. To test whether Kir2.1 was upregulated in SUR1-/- islets islet lysates were immunoblotted for Kir2.1 protein. Kir2.1 was expressed in mouse and human islets and increased in lysates from freshly isolated SUR1 islets. Using patch clamping, we found that an inwardly rectifying K current was present in freshly isolated SUR1-/- islets that was absent in wild type islets. This current was sensitive to the Kir2.1 inhibitors ML133 and barium. Notably, rectifying current, enhanced Kir2.1 protein levels, and the expected Kir2.1 drug sensitivity were all lost along with oscillations after overnight culture in media with 11.1 mM glucose. Similar results were found using fura-2 loaded islets to examine Ca2+ oscillations. To test whether the suppression of Kir2.1 activity in cultured islets was a response to culture in high glucose, islets were cultured in 5 mM instead of 11.1 mM glucose. Lowering glucose preserved Kir2.1 protein and maintained the islet oscillations. These results show that the loss of KATP channels produced by deleting SUR1 is at least partially compensated for by the upregulation of Kir2.1 channels in mouse beta cells. We propose pharmacological activation of compensatory Kir2.1 channels as a novel way to inhibit the unrestrained insulin secretion characteristic of beta cells from human CHI patients.

Up-Regulation of Kir2.1 (KCNJ2) by the Serum & Glucocorticoid Inducible SGK3

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 K_V11.1, which is expressed in cardiomyocytes. Cardiomyocytes further express the inward rectifier K⁺ channel K_ir2.1, which contributes to maintenance of resting cell membrane potential. Loss-of-function mutations of KCNJ2 encoding K_ir2.1 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_ir2.1. Methods: cRNA encoding K_ir2.1 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild type SGK3, constitutively active ^S419DSGK3 or inactive ^K191NSGK3. K_ir2.1 activity was determined by two-electrode voltage-clamp and K_ir2.1 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 ^S419DSGK3, but not ^K191NSGK3 significantly enhanced K_ir2.1-mediated currents. SGK inhibitor EMD638683 (50 µM) abrogated ^S419DSGK3-induced up-regulation of K_ir2.1. Moreover, wild type SGK3 enhanced the channel protein abundance in the cell membrane. The decay of K_ir2.1-mediated currents following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) was similar in oocytes coexpressing K_ir2.1 and SGK3 as in oocytes expressing K_ir2.1 alone, suggesting that SGK3 influences channel insertion into rather than channel retrieval from the cell membrane. Conclusions: SGK3 is a novel regulator of K_ir2.1.

Effects of intravitreal bevacizumab (Avastin) on the porcine retina

To evaluate the effects of intravitreal bevacizumab (Avastin) on the porcine retina, with respect to structural alterations, expression of proteins involved in apoptosis (bax, caspase-3, caspase-9) and gliosis (vimentin, GFAP), expression of factors which influence the development of vascular edema (VEGF, PEDF), and of membrane channels implicated in retinal osmohomeostasis (Kir4.1, aquaporin-1, aquaporin-4). One eye of seven adult pigs received a single intravitreal injection of bevacizumab (1.25mg). Control eyes received buffered saline. For light and electron microscopy, the eyes were prepared 3 (one animal) and 7 days (two animals) after injection. Retinal slices were immunostained against gliosis- and apoptosis-related proteins. The gene expression was determined in the neuroretina and the retinal pigment epithelium of the remaining four animals with real-time RT-PCR 2days after injection of bevacizumab. Intravitreal bevacizumab did not induce alterations in the retinal structure, neither at light microscopic nor at electron microscopic level. The photoreceptors were well-preserved; no signs of photoreceptor damage or mitochondrial swelling were observed. Bevacizumab did also not induce reactive gliosis (as indicated by the unaltered immunolocalization of the glial proteins vimentin, GFAP, and glutamine synthetase) or apoptosis (as indicated by the unaltered immunolocalization of bax, caspase-3, and caspase-9). Intravitreal bevacizumab decreased the transcriptional expression of VEGF-A, and increased the expression of Kir4.1 in the neuroretina and pigment epithelium, and of PEDF in the pigment epithelium. Bevacizumab did not alter the transcriptional expression of GFAP, bax, caspase-3, VEGF receptor-1 and -2, and aquaporin-1 and -4. A single intravitreal injection of bevacizumab does not result in structural changes of the porcine retina, nor in induction of gliosis or apoptosis. The bevacizumab-induced transcriptional downregulation of VEGF and upregulation of Kir4.1 might protect the retina from the development of vascular and cytotoxic edema.

Up-regulation of Kir2.1 by ER stress facilitates cell death of brain capillary endothelial cells

Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K+ channel (Kir2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of Kir channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca2+ concentration due to Ca2+ influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of Kir2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

Functional Characterization of Inward Rectifier Potassium Ion Channel in Murine Fetal Ventricular Cardiomyocytes

Aims: Previous studies have shown the dramatic changes in electrical properties of murine fetal cardiomyocytes, while details on inward rectifier potassium current (IK1) are still seldom discussed. Thus we aimed to characterize the functional expression and functional role of IK1 in murine fetal ventricular cardiomyocytes. Methods: Whole cell patch clamp was applied to investigate the electrophysiological properties of IK1. Quantitative real-time PCR, western blotting and double-label immunofluorescence were further utilized to find out the molecular basis of IK1. Results: Compared to early developmental stage (EDS), IK1 at late developmental stage (LDS) displayed higher current density, stronger rectifier property and faster activation kinetics. It was paralleled with the downregulation of Kir2.3 and the upregulation of Kir2.1/Kir2.2. IK1 contributed to maintain the maximum diastolic potential (MDP), late repolarization phase (LRP) as well as the action potential duration (APD). However, the contribution to MDP and velocity of LRP did not change significantly with maturation. Conclusions: During fetal development, the switch of IK1 subtypes from Kir2.1/Kir2.3 to Kir2.1 resulted in the dramatic changes in IK1 electrophysiological properties.

Analysis of Astroglial K+ Channel Expression in the Developing Hippocampus Reveals a Predominant Role of the Kir4.1 Subunit

Astrocytes in different brain regions display variable functional properties. In the hippocampus, astrocytes predominantly express time- and voltage-independent currents, but the underlying ion channels are not well defined. This ignorance is partly attributable to abundant intercellular coupling of these cells through gap junctions, impeding quantitative analyses of intrinsic membrane properties. Moreover, distinct types of cells with astroglial properties coexist in a given brain area, a finding that confused previous analyses. In the present study, we investigated expression of inwardly rectifying (Kir) and two-pore-domain (K2P) K+ channels in astrocytes, which are thought to be instrumental in the regulation of K+ homeostasis. Freshly isolated astrocytes were used to improve space-clamp conditions and allow for quantitative assessment of functional parameters. Patch-clamp recordings were combined with immunocytochemistry, Western blot analysis, and semiquantitative transcript analysis. Comparative measurements were performed in different CA1 subregions of astrocyte-targeted transgenic mice. While confirming weak Ba2+ sensitivity in situ, our data demonstrate that in freshly isolated astrocytes, the main proportion of membrane currents is sensitive to micromolar Ba2+ concentrations. Upregulation of Kir4.1 transcripts and protein during the first 10 postnatal days was accompanied by a fourfold increase in astrocyte inward current density. Hippocampal astrocytes from Kir4.1-/- mice lacked Ba2+-sensitive currents. In addition, we report functional expression of K2P channels of the TREK subfamily (TREK1, TREK2), which mediate astroglial outward currents. Together, our findings demonstrate that Kir4.1 constitutes the pivotal K+ channel subunit and that superposition of currents through Kir4.1 and TREK channels underlies the "passive" current pattern of hippocampal astrocytes.

Inward Rectifier Potassium Currents as a Target for Atrial Fibrillation Therapy

Subunits of inwardly rectifying potassium channels (Kir) are expressed in many different tissues of the human body. Inward rectifier currents expressed in the heart are constituted by pore-forming alpha-subunits of Kir2, Kir3, and Kir6 subfamilies. Characteristic properties of inward rectifiers comprise small outward conductances that nevertheless are important to terminal repolarization of cardiac action potentials. There is considerable difference in the regional expression of cardiac Kir channels, and subunits are additionally regulated by specific disease conditions. Resulting changes facilitate occurrence and persistence of atrial fibrillation (AF). For instance, upregulation of Kir2.1 protein and resultant current I K1 is a hallmark of AF-related ionic remodeling. Increased I K1 helps to stabilize atrial rotors, and current inhibition has accordingly been suggested as an antiarrhythmic approach for AF therapy. But there are caveats to I K1 inhibition per se, and there is no specific inhibitor of Kir2 channels. Modulation of I K1 rectification properties seems theoretically interesting for manipulation of Kir2 currents as an antiarrhythmic approach. Kir3-based muscarinic currents (I KACh) are functionally upregulated during AF through increased constitutive activity (passing current in the absence of an agonist). Upregulated I KACh supports sustenance of the arrhythmia. There is considerable intraatrial diversity in the expression of underlying Kir3.1/Kir3.4 subunits, but atrial-specific localization makes inhibition of this current a potentially interesting antiarrhythmic target devoid of ventricular side effects. Experimental studies of specific inhibitors indicate efficacy in various disease models. The role of I KATP remodeling under AF conditions has not been extensively studied, but present evidence indicates current downregulation and modulation of IKATP seems less promising than that of other inward rectifiers.

Initiation of human myoblast differentiation via dephosphorylation of Kir2.1 K+ channels at tyrosine 242

Myoblast differentiation is essential to skeletal muscle formation and repair. The earliest detectable event leading to human myoblast differentiation is an upregulation of Kir2.1 channel activity, which causes a negative shift (hyperpolarization) of the resting potential of myoblasts. After exploring various mechanisms, we found that this upregulation of Kir2.1 was due to dephosphorylation of the channel itself. Application of genistein, a tyrosine kinase inhibitor, increased Kir2.1 activity and triggered the differentiation process, whereas application of bpV(Phen), a tyrosine phosphatase inhibitor, had the opposite effects. We could show that increased Kir2.1 activity requires dephosphorylation of tyrosine 242; replacing this tyrosine in Kir2.1 by a phenylalanine abolished inhibition by bpV(Phen). Finally, we found that the level of tyrosine phosphorylation in endogenous Kir2.1 channels is considerably reduced during differentiation when compared with proliferation. We propose that Kir2.1 channels are already present at the membrane of proliferating, undifferentiated human myoblasts but in a silent state, and that Kir2.1 tyrosine 242 dephosphorylation triggers differentiation.

International Education Issues: Practicing and teaching international neurology

<h3>Background</h3> Dietary sodium intake regulates the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Whether the basolateral, inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1/Kir5.1) in the DCT is essential for mediating the effect of dietary sodium intake on NCC activity is unknown. <h3>Methods</h3> We used electrophysiology, renal clearance techniques, and immunoblotting to examine effects of Kir4.1/Kir5.1 in the DCT and NCC in wild-type and kidney-specific Kir4.1 knockout mice. <h3>Results</h3> Low sodium intake stimulated basolateral Kir4.1/Kir5.1 activity, increased basolateral K⁺ conductance, and hyperpolarized the membrane. Conversely, high sodium intake inhibited the potassium channel, decreased basolateral K⁺ currents, and depolarized the membrane. Low sodium intake increased total and phosphorylated NCC expression and augmented hydrochlorothiazide-induced natriuresis; high sodium intake had opposite effects. Thus, elevated NCC activity induced by low sodium intake was associated with upregulation of Kir4.1/Kir5.1 activity in the DCT, whereas inhibition of NCC activity by high sodium intake was associated with diminished Kir4.1/Kir5.1 activity. In contrast, dietary sodium intake did not affect NCC activity in knockout mice. Further, Kir4.1 deletion not only abolished basolateral K⁺ conductance and depolarized the DCT membrane, but also abrogated the stimulating effects induced by low sodium intake on basolateral K⁺ conductance and hyperpolarization. Finally, dietary sodium intake did not alter urinary potassium excretion rate in hypokalemic knockout and wild-type mice. <h3>Conclusions</h3> Stimulation of Kir4.1/Kir5.1 by low intake of dietary sodium is essential for NCC upregulation, and inhibition of Kir4.1/Kir5.1 induced by high sodium intake is a key step for downregulation of NCC.