Inward Rectifier Channels Research Articles

Structural changes in the conversion of an Arabidopsis outward-rectifying K+ channel into an inward-rectifying channel

Structural changes in the conversion of an Arabidopsis outward-rectifying K+ channel into an inward-rectifying channel

Lateral fenestrations in the extracellular domain of the glycine receptor contribute to the main chloride permeation pathway.

Glycine receptors (GlyRs) are ligand-gated ion channels mediating signal transduction at chemical synapses. Since the early patch-clamp electrophysiology studies, the details of the ion permeation mechanism have remained elusive. Here, we combine molecular dynamics simulations of a zebrafish GlyR-α1 model devoid of the intracellular domain with mutagenesis and single-channel electrophysiology of the full-length human GlyR-α1. We show that lateral fenestrations between subunits in the extracellular domain provide the main translocation pathway for chloride ions to enter/exit a central water-filled vestibule at the entrance of the transmembrane channel. In addition, we provide evidence that these fenestrations are at the origin of current rectification in known anomalous mutants and design de novo two inward-rectifying channels by introducing mutations within them. These results demonstrate the central role of lateral fenestrations on synaptic neurotransmission.

Effects of Ruanmailing in Blocking Early Stages of Atherosclerosis by TNF-α Regulation via Kir2.1.

Objective Ruanmailing oral solution consists of 16 herbs, has anti-lipid peroxidation activity, protects vascular endothelial cells, and improves vascular elasticity. It is an effective drug for the treatment of atherosclerosis (AS). The objective of this study was to investigate the mechanism underlying the antiatherosclerotic effects of Ruanmailing oral solution. Methods Macrophages were isolated, cultured, and divided into the macrophage control; macrophage foam cell; and low-, medium-, and high-concentration Ruanmailing groups. Cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay, and the expression levels of inward-rectifier potassium ion channel 2.1 (Kir2.1) and tumor necrosis factor (TNF)-α were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Results CCK-8 assay results showed that the tested concentrations of Ruanmailing solution did not affect macrophage proliferation. RT-PCR and Western blot assays indicated that TNF-α expression increased significantly with the formation of macrophage foam cells (P < 0.05). In addition, significant decreases in Kir2.1 and TNF-α expression were observed following treatment with various concentrations of Ruanmailing (P < 0.05). Conclusion Based on the results, Ruanmailing affects macrophage foam cell formation by regulating Kir2.1 expression, which in turn reduces TNF-α expression and exerts antiatherosclerotic effects. These findings provide a scientific basis for the use of traditional Chinese medicine for AS treatment.

Molecular structure of an open human KATP channel

KATP channels are metabolic sensors that translate intracellular ATP/ADP balance into membrane excitability. The molecular composition of KATP includes an inward-rectifier potassium channel (Kir) and an ABC transporter-like sulfonylurea receptor (SUR). Although structures of KATP have been determined in many conformations, in all cases, the pore in Kir is closed. Here, we describe human pancreatic KATP (hKATP) structures with an open pore at 3.1- to 4.0-Å resolution using single-particle cryo-electron microscopy (cryo-EM). Pore opening is associated with coordinated structural changes within the ATP-binding site and the channel gate in Kir. Conformational changes in SUR are also observed, resulting in an area reduction of contact surfaces between SUR and Kir. We also observe that pancreatic hKATP exhibits the unique (among inward-rectifier channels) property of PIP2-independent opening, which appears to be correlated with a docked cytoplasmic domain in the absence of PIP2.

A computational structural biology study to understand the impact of mutation on structure–function relationship of inward-rectifier potassium ion channel Kir6.2 in human

Type 2 diabetes (T2D) is clinically characterized via hyperglycemia. Polymorphism rs5219 in the KCNJ11 gene is a risk factor for developing T2D in humans. KCNJ11 encodes the ‘inward-rectifier potassium ion channel (Kir6.2)’. However, because of the absence of the complete crystal/NMR structures of Kir6.2 proteins, insight into its structure and function and its interaction with diverse ligands remain elusive to date. Therefore, a computational approach was employed for predicting the best plausible ‘three-dimensional’ structure of Kir6.2 as well as for studying the influence of mutation (p. GLU23LYS) on both architectures as well as the function of Kir6.2 employing simulation studies. Results obtained revealed that though, with increased time, ‘Gibbs free energy’ becomes positive, residues in wild type Kir6.2 experiences less random movement as compared to mutant Kir6.2. The less random movement of residues in wild type Kir6.2 represents the standard coupling between open and closing of ‘KATP channel’ and thus the normal secretion of insulin. The more dispersed motion of mutant Kir6.2 residues represents ‘overactivity’ of the ‘KATP channel’ and thus insulin ‘under-secretion’. Further, molecular docking and simulation studies identified two phytochemicals/drugs, namely, A-348441 and chushizisin I, which retains the wild type property of Kir6.2 after binding with mutant protein. Unlike A-348441, this is for the first time, the present study is reporting about the plausible anti-diabetic property of chushizisin I. As these two phytochemicals/drugs, namely, A-348441 and chushizisin I, have passed ADMET test, in the near future, they may be utilized as anti-diabetic drugs after further investigation. Communicated by Ramaswamy H. Sarma

Electrhopysiological Effect of the Polyamine Spermine in Normoxic and Ischemic Ventricular Myocardium

Cytoplasmic polyamines (PA) are involved in control of many cellular functions and are well known as regulators of so called inward-rectifierpotassium ion channels. Nevertheless, functional significance of extracellular PA in the heart is poorly elucidated. Aim of this study wasto study effects of endogenous PA spermine in the ventricular myocardium. Effects of the extracellular spermine were investigated in isolatedmulticellular preparations of rabbit and rat ventricular myocardium. Langendorff-perfused isolated rat and rabbit hearts were alsoused. Action potential (APs) duration and pattern of excitation in ventricular myocardium were estimated using standard microelectrodetechnique and optical mapping. Functional refractory periods were assessed in Langendorff perfused hearts with the help of programmedelectrical stimulation of the ventricle. In this study extracellular PA spermine (0.1-5 mM) induced shortening of the APs in multicellularpreparations of rat ventricular myocardium registered using sharp microelectrode technique. However, spermine caused only weak effectin preparations of ventricular myocardium from rabbit heart: highest tested concentration of spermine (5 mM) induced 4.7 % APsshortening. Similarly, 0.1-1 mM of spermine was unable to alter substantially ventricular effective refractory periods in isolated perfusedrabbit hearts. In two animal species tested (rat and rabbit) 0.1-1 mM of spermine failed to affect conduction velocity and activation patternin ventricles of isolated Langendorff-perfused hearts under normoxia. However, in the rat no-flow model of ischemia-reperfusion extracellularspermine improved conduction of excitation in ventricles. Our results allow suggesting that extracellular spermine can preventischemia-induced proarrhythmic changes in ventricular myocardium probably due to reduction of calcium accumulation, but this effectis significant only when PA is applied in millimolar concentrations. Also, potential anti-ischemic effect of the PA may be species specific.

Electrophysiological Identification and Activity Analyses of Plasma Membrane K+ Channels in Maize Guard Cells.

Stomatal movement, which plays an essential role in plant transpiration and photosynthesis, is controlled by ion channels that mediate K+ and anion fluxes across the plasma membrane (PM) of guard cells. These channels in dicots are accurately regulated by various physiological factors, such as pH, abscisic acid (ABA) and Ca2+; however, the data in monocots are limited. Here the whole-cell patch-clamping technique was applied to analyze the properties and regulations of PM K+ channels in maize guard cells. The results indicated that the hyperpolarization-activated inward-rectifying channels were highly K+-selective. These inward K+ (Kin) channels were sensitive to extracellular K+. Their slope factor (S) decreased when the apoplastic K+ concentration decline, causing a positive shift of the half-activation potential (V1/2). Their activities were promoted by apoplastic acidification but inhibited by apoplastic and cytosolic alkalization. Nevertheless, the outward K+ (Kout) channel activities were uniquely promoted by cytosolic alkalization. Both apoplastic and cytosolic ABA inhibited Kin channels independent of cytosolic Ca2+ ([Ca2+]cyt). And two Ca2+-dependent mechanisms with different Ca2+ affinities may mediate resting- and high-[Ca2+]cyt-induced inhibition on Kin channels, respectively. However, resting [Ca2+]cyt impaired the inhibition of Kin channels induced by apoplastic ABA, not cytosolic ABA. Furthermore, the result that high [Ca2+]cyt attenuated ABA-induced inhibition highlighted the importance of [Ca2+]cyt for Kin channel regulation. There may exist a Ca2+-dependent regulation of the Ca2+-independent ABA signaling pathways for Kin channel inhibition. These results provided an electrophysiological view of the multiple level regulations of PM K+ channel activities and kinetics in maize guard cells.

Intercellular Connectivity and Multicellular Bioelectric Oscillations in Nonexcitable Cells: A Biophysical Model.

Bioelectricity is emerging as a crucial mechanism for signal transmission and processing from the single-cell level to multicellular domains. We explore theoretically the oscillatory dynamics that result from the coupling between the genetic and bioelectric descriptions of nonexcitable cells in multicellular ensembles, connecting the genetic prepatterns defined over the ensemble with the resulting spatio-temporal map of cell potentials. These prepatterns assume the existence of a small patch in the ensemble with locally low values of the genetic rate constants that produce a specific ion channel protein whose conductance promotes the cell-polarized state (inward-rectifying channel). In this way, the short-range interactions of the cells within the patch favor the depolarized membrane potential state, whereas the long-range interaction of the patch with the rest of the ensemble promotes the polarized state. The coupling between the local and long-range bioelectric signals allows a binary control of the patch membrane potentials, and alternating cell polarization and depolarization states can be maintained for optimal windows of the number of cells and the intercellular connectivity in the patch. The oscillatory phenomena emerge when the feedback between the single-cell bioelectric and genetic dynamics is coupled at the multicellular level. In this way, the intercellular connectivity acts as a regulatory mechanism for the bioelectrical oscillations. The simulation results are qualitatively discussed in the context of recent experimental studies.

Change of voltage-gated potassium channel 1.7 expressions in monocrotaline-induced pulmonary arterial hypertension rat model.

PurposeAbnormal potassium channels expression affects vessel function, including vascular tone and proliferation rate. Diverse potassium channels, including voltage-gated potassium (Kv) channels, are involved in pathological changes of pulmonary arterial hypertension (PAH). Since the role of the Kv1.7 channel in PAH has not been previously studied, we investigated whether Kv1.7 channel expression changes in the lung tissue of a monocrotaline (MCT)-induced PAH rat model and whether this change is influenced by the endothelin (ET)-1 and reactive oxygen species (ROS) pathways.MethodsRats were separated into 2 groups: the control (C) group and the MCT (M) group (60 mg/kg MCT). A hemodynamic study was performed by catheterization into the external jugular vein to estimate the right ventricular pressure (RVP), and pathological changes in the lung tissue were investigated. Changes in protein and mRNA levels were confirmed by western blot and polymerase chain reaction analysis, respectively.ResultsMCT caused increased RVP, medial wall thickening of the pulmonary arterioles, and increased expression level of ET-1, ET receptor A, and NADPH oxidase (NOX) 4 proteins. Decreased Kv1.7 channel expression was detected in the lung tissue. Inward-rectifier channel 6.1 expression in the lung tissue also increased. We confirmed that ET-1 increased NOX4 level and decreased glutathione peroxidase-1 level in pulmonary artery smooth muscle cells (PASMCs). ET-1 increased ROS level in PASMCs.ConclusionDecreased Kv1.7 channel expression might be caused by the ET-1 and ROS pathways and contributes to MCT-induced PAH.

Abstract 432: MCUB Regulates Mitochondrial Calcium Uniporter Channel Gating and Modulates Bioenergetics and Cell Death

The mitochondrial calcium uniporter (MCU) is a high-capacity, inward-rectifying channel in the inner mitochondrial membrane and is required for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. m Ca 2+ signaling regulates bioenergetics and activates the mitochondrial permeability transition pore (MPTP) which are cellular processes implicated in cardiac pathophysiology warranting further research into the molecular regulation of the MCU. Recently, a MCU gene paralog, MCUB , was identified as a possible component of the channel. To investigate MCUB’s contribution to uniporter regulation we created a MCUB -/- HeLa cell line using CRISPR-Cas9n. Here, we report that loss of MCUB increased m Ca 2+ transient amplitude after IP3R stimulation (52% vs. con) suggesting MCUB negatively regulates m Ca 2+ uptake. Mitoplast patch-clamping confirmed that loss of MCUB increases MCU current density, suggesting MCUb modulates channel capacitance. Permeabilized MCUB -/- and WT cells exposed to various levels of Ca 2+ (0.5-20μM) revealed that MCUB -/- cells exhibited m Ca 2+ uptake at lower Ca 2+ concentrations than controls, suggesting MCUB contributes to channel gating. In m Ca 2+ retention capacity experiments MCUB -/- cells required ~30% less bath Ca 2+ to induce depolarization, suggesting a predisposition to m Ca 2+ overload. Next, we generated a cardiac-specific, tamoxifen-inducible MCUB overexpression mouse model ( MCUB -Tg). Cardiomyocytes isolated from MCUB -Tg hearts exhibited decreased m Ca 2+ uptake at low-Ca 2+ (59% vs. con) and isolated mitochondria exhibited a reduction in Ca 2+ -induced swelling (37% vs. con), suggesting a resistance to permeability transition. MCUB -Tg mice displayed a significant impairment in isoproterenol-induced contractile reserve and this correlated with a loss of isoproterenol-mediated activation of pyruvate dehydrogenase. In summary, our results suggest that MCUB limits m Ca 2+ uptake by altering channel-gating and thereby regulates bioenergetics and MPTP opening.

The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

Calbindin-D9k Ablation Disrupt Glucose/Pancreatic Insulin Homeostasis

It has been proposed that cellular Ca2+ signals activate hormone secretion. In pancreatic β cells, which produce insulin, Ca2+ signals have been known to contribute to insulin secretion. Prior to this study, we confirmed that insulin-secreting β cells express CaBP-9k, and assumed that CaBP-9k play a role in β cell insulin synthesis or secretion. Using CaBP-9k knock out (KO) mice, we demonstrated that ablation of CaBP-9k causes reducing insulin secretion and increasing serum glucose. To compare the role of CaBP-9k with pathophysiological conditions, we exposed wild-type and CaBP-9k KO mice to hypoxic conditions for 10 days. Hypoxia induced endoplasmic reticulum (ER) stress, increasing both insulin signaling and insulin resistance. By exposing hypoxia, CaBP-9k KO mice showed an increased level of ER stress marker protein relative to wild type mice. Without hypoxic conditions, CaBP-9K ablation regulates calcium channels and causes ER stress in a CaBP-9K specific manner. Ablation of CaBP-9k also showed decreased levels of sulfonylurea receptor1 (SUR1) and inward-rectifier potassium ion channel 6.2 (Kir6.2), which are insulin secretion marker genes. Overall, the results of the present study demonstrated that CaBP-9k regulates synthesis of insulin and is part of the insulin-secreting calcium signaling.

Monogenic Diabetes: What It Teaches Us on the Common Forms of Type 1 and Type 2 Diabetes.

To date, more than 30 genes have been linked to monogenic diabetes. Candidate gene and genome-wide association studies have identified > 50 susceptibility loci for common type 1 diabetes (T1D) and approximately 100 susceptibility loci for type 2 diabetes (T2D). About 1-5% of all cases of diabetes result from single-gene mutations and are called monogenic diabetes. Here, we review the pathophysiological basis of the role of monogenic diabetes genes that have also been found to be associated with common T1D and/or T2D. Variants of approximately one-third of monogenic diabetes genes are associated with T2D, but not T1D. Two of the T2D-associated monogenic diabetes genes-potassium inward-rectifying channel, subfamily J, member 11 (KCNJ11), which controls glucose-stimulated insulin secretion in the β-cell; and peroxisome proliferator-activated receptor γ (PPARG), which impacts multiple tissue targets in relation to inflammation and insulin sensitivity-have been developed as major antidiabetic drug targets. Another monogenic diabetes gene, the preproinsulin gene (INS), is unique in that INS mutations can cause hyperinsulinemia, hyperproinsulinemia, neonatal diabetes mellitus, one type of maturity-onset diabetes of the young (MODY10), and autoantibody-negative T1D. Dominant heterozygous INS mutations are the second most common cause of permanent neonatal diabetes. Moreover, INS gene variants are strongly associated with common T1D (type 1a), but inconsistently with T2D. Variants of the monogenic diabetes gene Gli-similar 3 (GLIS3) are associated with both T1D and T2D. GLIS3 is a key transcription factor in insulin production and β-cell differentiation during embryonic development, which perturbation forms the basis of monogenic diabetes as well as its association with T1D. GLIS3 is also required for compensatory β-cell proliferation in adults; impairment of this function predisposes to T2D. Thus, monogenic forms of diabetes are invaluable "human models" that have contributed to our understanding of the pathophysiological basis of common T1D and T2D.

Effects of extracellular K+ on grapevine membrane potential as influenced by the antiviral mycophenolic acid. An electrophysiological study

Mycophenolic acid (MPA) is an effective antiviral drug in plants, and its action in modulating the activity of KATP channels is already known in animals. In the present work, an electrophysiological study was carried out to investigate MPA effects on plant K+ channels, through the measurement of trans-plasma membrane potential in samples of Vitis vinifera cv. Sangiovese treated with extracellular K+. Tests confirmed that the administration of MPA (in preincubated samples or in those maintained under chemical treatment) can reduce the membrane depolarization induced by K+. However, MPA-induced alteration in membrane potential was sensitive to the KATP channel opener diazoxide, as well to treatments with guanosine. This result confirms the effectiveness of MPA in influencing KATP channel activity as well as inhibiting activity of the inward-rectifier potassium ion channel which could be mediated by guanosine depletion induced by MPA.

Abstract 113: Renal Phenotype of Inwardly Rectifying Potassium Channel Kcnj16 (Kir 5.1) Knockout in the Dahl Salt-Sensitive Rats

The inward-rectifying channels play an important role in the control of resting membrane potential and tubular homeostasis in the kidney. Kcnj16 (Kir 5.1) form a heteromeric channel with Kcnj10 (Kir 4.1) at the basolateral membranes of aldosterone-sensitive distal nephron (ASDN); mutations in the human KCNJ10 gene result in SeSAME)/EAST syndrome, a complex disorder that includes salt wasting and hypokalemic alkalosis. To illuminate the importance of Kcnj16 (Kir 5.1) in the context of a disease state in vivo, we generated a Kcnj16 knockout rat model in Dahl salt-sensitive (SS) background by using ZFN technology. ZFN against Kcnj16 caused a 18-bp in-frame deletion that occurred in the second protein transmembrane domain. IHC analysis demonstrated highly specific expression of Kcnj16 on the basolateral membranes of ASDN in the control kidneys of SS rats, which was completely abolished in Kcnj16-/- rats. The electrophysiological recording of K+ channels in the CCD basolateral membrane revealed activity of only homomeric Kcnj10 channels (21 pS channel in Kcnj16-/- rats compared to both 41 and 21 pS channels in SS rats). Thus, these data provide evidence of successful knock out of this protein and consequent degradation of the channel in renal tubules. The Kcnj16-/- knockout in SS rat induces electrolyte imbalance, epileptic seizures and result in changes in development (37% reduction in body and 54% in kidney mass). The mean arterial pressure was significantly lower in Kcnj16-/- compared to SS rats (91.3±1.8 to 104.7±5.5 mmHg) when animals were fed a low salt (0.4%) diet. Knockout of Kcnj16 resulted in hypokalemia (4.25±0.09 vs 2.08±0.12 mmol/L in serum of control vs KO rats), hypermagnesemia (0.49±0.02 vs 0.63±0.01 mmol/L in serum of control vs KO rats), and FSGS. Urea electrolyte balance was also disturbed compared to control animals. Importantly, change of the diet to high salt (4%) caused mortality of KO rats within 1-2 days. These data demonstrate critical role of Kcnj16 channels in renal salt handling and in the development of salt-sensitive hypertension.

Characteristics of Na+ uptake in sugar beet (Beta vulgaris L.) seedlings under mild salt conditions

Salinity is one of the major environmental factors, which limits crop productivity worldwide. To investigate sodium (Na+) uptake pathways in sugar beet (Beta vulgaris L.) under mild salt conditions, in the present work, Na+ and potassium (K+) accumulation, Na+/K+ ratio, and Na+ and K+ net uptake rate in plants exposed to various concentrations of NaCl (0–50 mM) were analyzed in the absence or presence of KCl (10 and 50 mM) and K+ channel inhibitors Tetraethylammonium-Cl (TEA+, 5 and 10 mM), CsCl (Cs+, 3 and 6 mM) and BaCl2 (Ba2+, 5 and 10 mM). The results showed that high concentration (50 mM) of KCl significantly reduced Na+/K+ ratios in shoot and root of sugar beet in the absence or presence of NaCl. 10 or 50 mM KCl also decreased Na+ net uptake rate, or had no effects on it at 5, 10, and 50 mM NaCl, while enhanced K+ net uptake rate with external NaCl concentration at 5 and 25 mM. It seemed that high external K+ levels could maintain lower Na+/K+ ratio in sugar beet by enhancing K+ uptake and restricting Na+ uptake. Both 5 and 10 mM TEA+, which are considered to be a blocker of K+ channels, had no significant effects on net uptake rates of Na+ and K+ in sugar beet in the absence or presence of NaCl. However, 3 or 6 mM Cs+, which is also known to be an inhibitor of the K+ inward-rectifying channel (AKT1), led to significant reduction of K+ net uptake rate but did not affect Na+ net uptake rate in the presence of NaCl. 5 or 10 mM Ba2+, which is known as another blocker of K+ channel and transporter (HKT), not only reduced Na+ net uptake rate but also decreased K+ net uptake rate (except at 25 mM NaCl) in sugar beet at 5–50 mM NaCl. It is clear that Na+ uptake in sugar beet is very sensitive to Ba2+ but insensitive to TEA+ or Cs+, and that K+ uptake is sensitive to Cs+ or Ba2+, whereas it is insensitive to TEA+. We proposed that the AKT1 may mediate K+ uptake and HKT1 may mediate Na+ uptake in sugar beet at 5–50 mM NaCl.

Nitrite as Direct S-Nitrosylating Agent of Kir2.1 Channels.

Nitrite, a physiological nitric oxide (NO) storage form and an alternative way for NO generation, affects numerous biological processes through NO-dependent and independent pathways, including the S-nitrosylation of thiol-containing proteins. Mechanisms underlying these phenomena are not fully understood. The purpose of this study was to analyse in the rat heart (as prototype of mammalian heart) whether nitrite affects S-nitrosylation of cardiac proteins and the potential targets for S-nitrosylation. Rat hearts, perfused according to Langendorff, were exposed to nitrite. By Biotin Switch Method, we showed that nitrite treatment increased the degree of S-nitrosylation of a broad range of membrane proteins. Further analysis, conducted on subfractioned proteins, allowed us to identify a high level of nitrosylation in a small range of plasmalemmal proteins characterized by using an anti-Kir2.1 rabbit polyclonal antibody. We also verified that this effect of nitrite is preserved in the presence of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide). Our results suggest, for the first time, that nitrite represents a direct S-nitrosylating agent in cardiac tissues and that inward-rectifier potassium ion channels (Kir2.1) are one of the targets. These observations are of relevance since they support the growing evidence that nitrite is not only a NO reserve but also a direct modulator of important functional cardiac proteins.

Renal phenotype of inwardly rectifying potassium channel Kcnj16 (Kir 5.1) knockout in the Dahl salt‐sensitive rats (893.16)

The inward‐rectifying channels play an important role in the control of resting membrane potential and tubular homeostasis in the kidney. To illuminate the importance of Kcnj16 (Kir 5.1) in the context of a disease state in vivo, we generated a Kcnj16 knockout rat model in Dahl salt‐sensitive (SS) background by using zinc finger nuclease (ZFN) technology. ZFN against Kcnj16 caused a 18‐bp in‐frame deletion that occurred in the second protein transmembrane domain. IHC analysis demonstrated highly specific expression of Kcnj16 on the basolateral membranes of cortical collecting ducts and distal convoluted tubules in the control kidneys of SS and Sprague Dawley rats, which was completely abolished in Kcnj16‐/‐ rats; thus, successful knock out of this protein and consequent degradation of the channel in renal tubules were shown. The mean arterial pressure was significantly lower in Kcnj16‐/‐ compared to wild type SS rats (91.3±1.8 to 104.7±5.5 mmHg) when animals were fed a low salt (0.4%) diet. BUN, potassium and magnesium plasma concentrations were significantly modulated in knock out rats. Urea electrolyte balance was also disturbed compared to wild type animals. Importantly, change of the diet to high salt (4%) chow resulted in mortality of KO rats within 1‐2 days. These data demonstrate critical role of Kcnj16 channels in renal salt handling and in the development of salt‐sensitive hypertension.Grant Funding Source: Supported by NIH HL108880 and ADA 1‐10‐BS‐168 to A.S., NHLBI 5RC2HL101681 to H.J.J

KCNJ11: Genetic Polymorphisms and Risk of Diabetes Mellitus

Diabetes mellitus (DM) is a major worldwide health problem and its prevalence has been rapidly increasing in the last century. It is caused by defects in insulin secretion or insulin action or both, leading to hyperglycemia. Of the various types of DM, type 2 occurs most frequently. Multiple genes and their interactions are involved in the insulin secretion pathway. Insulin secretion is mediated through the ATP-sensitive potassium (KATP) channel in pancreatic beta cells. This channel is a heteromeric protein, composed of four inward-rectifier potassium ion channel (Kir6.2) tetramers, which form the pore of the KATP channel, as well as sulfonylurea receptor 1 subunits surrounding the pore. Kir6.2 is encoded by the potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11) gene, a member of the potassium channel genes. Numerous studies have reported the involvement of single nucleotide polymorphisms of the KCNJ11 gene and their interactions in the susceptibility to DM. This review discusses the current evidence for the contribution of common KCNJ11 genetic variants to the development of DM. Future studies should concentrate on understanding the exact role played by these risk variants in the development of DM.

Transforming growth factor‐β1 primes proliferating adult neural progenitor cells to electrophysiological functionality

The differentiation of adult neural progenitors (NPCs) into functional neurons is still a limiting factor in the neural stem cell field but mandatory for the potential use of NPCs in therapeutic approaches. Neuronal function requires the appropriate electrophysiological properties. Here, we demonstrate that priming of NPCs using transforming growth factor (TGF)-β1 under conditions that usually favor NPCs' proliferation induces electrophysiological neuronal properties in adult NPCs. Gene chip array analyses revealed upregulation of voltage-dependent ion channel subunits (Kcnd3, Scn1b, Cacng4, and Accn1), neurotransmitters, and synaptic proteins (Cadps, Snap25, Grik4, Gria3, Syngr3, and Gria4) as well as other neuronal proteins (doublecortin [DCX], Nrxn1, Sept8, and Als2cr3). Patch-clamp analysis demonstrated that control-treated cells expressed only voltage-dependent K(+) -channels of the delayed-rectifier type and the A-type channels. TGF-β1-treated cells possessed more negative resting potentials than nontreated cells owing to the presence of delayed-rectifier and inward-rectifier channels. Furthermore, TGF-β1-treated cells expressed voltage-dependent, TTX-sensitive Na(+) channels, which showed increasing current density with TGF-β1 treatment duration and voltage-dependent (+)BayK8644-sensitive L-Type Ca(2+) channels. In contrast to nontreated cells, TGF-β1-treated cells responded to current injections with action-potentials in the current-clamp mode. Furthermore, TGF-β1-treated cells responded to application of GABA with an increase in membrane conductance and showed spontaneous synaptic currents that were blocked by the GABA-receptor antagonist picrotoxine. Only NPCs, which were treated with TGF-β1, showed Na(+) channel currents, action potentials, and GABAergic currents. In summary, stimulation of NPCs by TGF-β1 fosters a functional neuronal phenotype, which will be of relevance for future cell replacement strategies in neurodegenerative diseases or acute CNS lesions.