Intracellular Potassium Concentration Research Articles

Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis.

The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K+ and Cl− efflux via activation of K+ channels, volume-regulated anion channels (VRACs), and the K+-Cl− cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na+-K+-2Cl− cotransporter isoform 1 (NKCC1). This results in a rapid (<10 min) and significant (>90%) reduction in intracellular K+ content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.

A jack of all trades: the multiple roles of the unique essential second messenger cyclic di-AMP.

Second messengers are key components of many signal transduction pathways. In addition to cyclic AMP, ppGpp and cyclic di-GMP, many bacteria use also cyclic di-AMP as a second messenger. This molecule is synthesized by distinct classes of diadenylate cyclases and degraded by phosphodiesterases. The control of the intracellular c-di-AMP pool is very important since both a lack of this molecule and its accumulation can inhibit growth of the bacteria. In many firmicutes, c-di-AMP is essential, making it the only known essential second messenger. Cyclic di-AMP is implicated in a variety of functions in the cell, including cell wall metabolism, potassium homeostasis, DNA repair and the control of gene expression. To understand the molecular mechanisms behind these functions, targets of c-di-AMP have been identified and characterized. Interestingly, c-di-AMP can bind both proteins and RNA molecules. Several proteins that interact with c-di-AMP are required to control the intracellular potassium concentration. In Bacillus subtilis, c-di-AMP also binds a riboswitch that controls the expression of a potassium transporter. Thus, c-di-AMP is the only known second messenger that controls a biological process by interacting with both a protein and the riboswitch that regulates its expression. Moreover, in Listeria monocytogenes c-di-AMP controls the activity of pyruvate carboxylase, an enzyme that is required to replenish the citric acid cycle. Here, we review the components of the c-di-AMP signaling system.

Neurologic Consequences of Electrolyte Disorders

Characteristically, several fluid and electrolyte disorders have dysfunction of the central nervous system (CNS) as a major complication. The brain, within a closed space, cannot respond quickly or effectively to prevent the acute stresses of osmotic swelling or contraction of extracellular and/or intracellular spaces. Disorders of many other ions and neutral molecules can lead to pathology, including cognitive abnormalities, encephalopathy, falls, and seizures. This chapter looks at osmolar change and brain volume regulation and the importance of sodium (Na), potassium, phosphate, and magnesium. The section on sodium looks at its importance to serum osmolality and discusses hyponatremia and hypernatremia, syndrome of inappropriate antidiuretic hormone (SIADH) and cerebral salt wasting, dialysis disequilibrium syndrome, and the CNS effects of elevated ammonia and altered glucose levels. The section on potassium discusses the ratio between the intracellular and extracellular potassium concentrations and looks at hypokalemia and hyperkalemia. Phosphate plays a vital role in energy metabolism, and this section explores hypercalcemia and hypocalcemia. Magnesium’s role in cell proliferation and energy metabolism is discussed, with subsections on hypomagnesemia and hypermagnesemia. Tables list symptoms of electrolyte disorders, ways to distinguish between SIADH and cerebral salt wasting, risk factors for osmotic demyelination, and expansion and contraction syndromes. Figures include a graph showing serum Na against urine osmolality in a patient with symptomatic hyponatremia, and a chart of a woman with hyperammonemia and severe metabolic acidosis. This chapter contains 2 highly rendered figures, 4 tables, 65 references, 5 MCQs.

Neurologic Consequences of Electrolyte Disorders

Characteristically, several fluid and electrolyte disorders have dysfunction of the central nervous system (CNS) as a major complication. The brain, within a closed space, cannot respond quickly or effectively to prevent the acute stresses of osmotic swelling or contraction of extracellular and/or intracellular spaces. Disorders of many other ions and neutral molecules can lead to pathology, including cognitive abnormalities, encephalopathy, falls, and seizures. This chapter looks at osmolar change and brain volume regulation and the importance of sodium (Na), potassium, phosphate, and magnesium. The section on sodium looks at its importance to serum osmolality and discusses hyponatremia and hypernatremia, syndrome of inappropriate antidiuretic hormone (SIADH) and cerebral salt wasting, dialysis disequilibrium syndrome, and the CNS effects of elevated ammonia and altered glucose levels. The section on potassium discusses the ratio between the intracellular and extracellular potassium concentrations and looks at hypokalemia and hyperkalemia. Phosphate plays a vital role in energy metabolism, and this section explores hypercalcemia and hypocalcemia. Magnesium’s role in cell proliferation and energy metabolism is discussed, with subsections on hypomagnesemia and hypermagnesemia. Tables list symptoms of electrolyte disorders, ways to distinguish between SIADH and cerebral salt wasting, risk factors for osmotic demyelination, and expansion and contraction syndromes. Figures include a graph showing serum Na against urine osmolality in a patient with symptomatic hyponatremia, and a chart of a woman with hyperammonemia and severe metabolic acidosis. This chapter contains 2 highly rendered figures, 4 tables, 65 references, 5 MCQs.

Minimum conditions for the induction of cortical spreading depression in brain slices.

Cortical spreading depression (CSD) occurs during various forms of brain injury such as stroke, subarachnoid hemorrhage, and brain trauma, but it is also thought to be the mechanism of the migraine aura. It is therefore expected to occur over a range of conditions including the awake behaving state. Yet it is unclear how such a massive depolarization could occur under relatively benign conditions. Using a microfluidic device with focal stimulation capability in a mouse brain slice model, we varied extracellular potassium concentration as well as the area exposed to increased extracellular potassium to determine the minimum conditions necessary to elicit CSD. Importantly, we focused on potassium levels that are physiologically plausible (≤145 mM; the intracellular potassium concentration). We found a strong correlation between the threshold concentration and the slice area exposed to increased extracellular potassium: minimum area of exposure was needed with the highest potassium concentration, while larger areas were needed at lower concentrations. We also found that moderate elevations of extracellular potassium were able to elicit CSD in relatively small estimated tissue volumes that might be activated under noninjury conditions. Our results thus show that CSD may be inducible under the conditions that expected in migraine aura as well as those related to brain trauma.

Transepithelial glucose transport and Na+/K+ homeostasis in enterocytes: an integrative model

The uptake of glucose and the nutrient coupled transcellular sodium traffic across epithelial cells in the small intestine has been an ongoing topic in physiological research for over half a century. Driving the uptake of nutrients like glucose, enterocytes must have regulatory mechanisms that respond to the considerable changes in the inflow of sodium during absorption. The Na-K-ATPase membrane protein plays a major role in this regulation. We propose the hypothesis that the amount of active Na-K-ATPase in enterocytes is directly regulated by the concentration of intracellular Na(+) and that this regulation together with a regulation of basolateral K permeability by intracellular ATP gives the enterocyte the ability to maintain ionic Na(+)/K(+) homeostasis. To explore these regulatory mechanisms, we present a mathematical model of the sodium coupled uptake of glucose in epithelial enterocytes. Our model integrates knowledge about individual transporter proteins including apical SGLT1, basolateral Na-K-ATPase, and GLUT2, together with diffusion and membrane potentials. The intracellular concentrations of glucose, sodium, potassium, and chloride are modeled by nonlinear differential equations, and molecular flows are calculated based on experimental kinetic data from the literature, including substrate saturation, product inhibition, and modulation by membrane potential. Simulation results of the model without the addition of regulatory mechanisms fit well with published short-term observations, including cell depolarization and increased concentration of intracellular glucose and sodium during increased concentration of luminal glucose/sodium. Adding regulatory mechanisms for regulation of Na-K-ATPase and K permeability to the model show that our hypothesis predicts observed long-term ionic homeostasis.

Quantification of potassium levels in cells treated with Bordetella adenylate cyclase toxin

The aim of this study was to compare two methods for quantification of changes in intracellular potassium concentration (decrease from ∼140 to ∼20mM) due to the action of a pore-forming toxin, the adenylate cyclase toxin (CyaA) from the pathogenic bacterium Bordetella pertussis. CyaA was incubated with stably transfected K1 Chinese hamster ovary cells expressing the toxin receptor CD11b/CD18 and the decrease in potassium concentration in the cells was followed by inductively coupled plasma mass spectrometry (ICP–MS). It is shown that this method is superior in terms of sensitivity, accuracy, and temporal resolution over the method employing the potassium-binding benzofuran isophthalate–acetoxymethyl ester fluorescent indicator. The ICP–MS procedure was found to be a reliable and straightforward analytical approach enabling kinetic studies of CyaA action at physiologically relevant toxin concentrations (<1000ng/ml) in biological microsamples.

Understanding of effects of potassium on cardiac tissue by medical students: a critical appraisal

Potassium (K + ) is one of the most important ion present in the human body and involved in numerous physiological activities. It mainly affects heart and skeletal muscle but the effects are not confined to theses organs only. The article precisely focuses on the explaining the physiological as well as pathological aspects of potassium on cardiac tissue. This article tends to explain: The cause of difference in extra cellular and Intra cellular concentration of potassium when potassium channels are open in resting conditions, why are Purkinje fibers and ectopic tissue are more sensitive to effect of potassium, mechanisms responsible for increased action potential duration by hypokalemia and decreased action potential duration by hyperkalemia. Hypokalemia generates ectopic activities and hyperkalemia inhibits them, therapeutic effects of potassium administration without causing hyperkalemia. These issues will be discussed and try to be explained with the help of Ohm’s law , Nernst equation and Nernst potential to sort out the complicated actions of potassium on cardiac tissue in a simplified manner. The primary aim of article is to improve understanding of potassium physiology by medical graduates, secondarily, convey message regarding improvement in teaching methodology in Pharmacology for the benefit of new generations.

Potassium uptake system Trk2 is crucial for yeast cell viability during anhydrobiosis

Yeasts grow at very different potassium concentrations, adapting their intracellular cation levels to changes in the external environment. Potassium homeostasis is maintained with the help of several transporters mediating the uptake and efflux of potassium with various affinities and mechanisms. In the model yeast Saccharomyces cerevisiae, two uptake systems, Trk1 and Trk2, are responsible for the accumulation of a relatively high intracellular potassium content (200-300mM) and the efflux of surplus potassium is mediated by the Tok1 channel and active exporters Ena ATPase and Nha1 cation/proton antiporter. Using a series of deletion mutants, we studied the role of individual potassium transporters in yeast cell resistance to dehydration. The Trk2 transporter (whose role in S.cerevisiae physiology was not clear) is important for cell viability in the stationary phase of growth and, moreover, it plays a crucial role in the yeast survival of dehydration/rehydration treatments. Mutants lacking the TRK2 gene accumulated significantly lower amounts of potassium ions in the stationary culture growth phase, and these lower amounts correlated with decreased resistance to dehydration/rehydration stress. Our results showed Trk2 to be the major potassium uptake system in stationary cells, and potassium content to be a crucial parameter for desiccation survival.

Pathogenetic role of magnesium deficiency in ophthalmic diseases.

Magnesium is one of the most important regulatory cation involved in several biological processes. It is important for maintaining the structural and functional integrity of several vital ocular tissues such as cornea, lens and retina. The magnesium content of lens, especially in its peripheral part, is higher than that in aqueous and vitreous humor. Magnesium has also been shown to play critically important role in retinal functions. Magnesium plays significant role as a cofactor for more than 350 enzymes in the body andregulates neuroexcitability and several ion channels. Membrane associated ATPase functions that are crucial in regulating the intracellular ionic environment, are magnesium-dependent. Moreover, the enzymes involved in ATP production and hydrolysis are also magnesium-dependent. Magnesium deficiency by interfering with ATPase functions causes increased intracellular calcium and sodium and decreases intracellular potassium concentration. Such ionic imbalances in turn alter the other cellular enzymatic reactions and form the basis of the association of magnesium deficiency with ophthalmic diseases such as cataract. In presence of magnesium deficiency, an imbalance between mediators of vasoconstriction and vasorelaxation may underlie the vasospasm, which is one of the pathogenic factors in primary open angle glaucoma. Furthermore, magnesium deficiency is also a contributing factor in increased oxidative stress and inducible NOS stimulation that can further contribute in the initiation and progression of ocular pathologies such as cataract, glaucoma and diabetic retinopathy. In this paper we review the association of disturbances of magnesium homeostasis with several ophthalmic diseases.

Hypokalemia and Hyperkalemia in Infants and Children: Pathophysiology and Treatment

Potassium is the second most abundant cation in the body. About 98% of potassium is intracellular and that is particularly in the skeletal muscle. Electrical disturbances associated with disorders of potassium homeostasis are a function of both the extracellular and intracellular potassium concentrations. Clinical disorders of potassium homeostasis occur with some regularity, especially in hospitalized patients receiving many medications. This article will review the pathophysiology of potassium homeostasis, symptoms, causes, and treatment of hypo- and hyperkalemia.

Dengue Virus Capsid Protein Binding to Lipid Droplets and its Inhibition. towards a New Drug Target

Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths yearly. No effective treatment is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for viral replication. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed with DENV C-functionalized AFM tips. DENV C-LDs interaction was found to be strong and dependent on the high intracellular potassium concentration. Inhibition of Na+/K+-ATPase in DENV-infected cells resulted in the dissociation of DENV C from LDs and a 50-fold inhibition of infectious virus production (but not of RNA replication), demonstrating the biological relevance of the K+-dependence. Limited LDs proteolysis impaired DENV C-LDs interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C ligand on the LDs surface. A peptide based on a conserved segment of DENV C intrinsically disordered N-terminal domain (pep14-23) was shown by force spectroscopy to successfully inhibit DENV C-LDs interaction. These advances pave the way to drug development approaches, in which inhibitory efficiency may be improved through lead optimization. A similar strategy may be used for other flaviviruses.Carvalho et al. (2012) J. Virol., 86, 2096-2108.Martins et al. (2012) Biochem. J., 444, 405-415.

Osmotic tolerance and intracellular ion concentrations of bovine sperm are affected by cryopreservation

In this study, the effects of cryopreservation on osmoregulation and ion homeostasis in bovine sperm were studied. We determined: (1) the osmotic tolerance limits and cell volume response upon exposure to anisotonic conditions, (2) the intracellular pH and potassium concentration, and (3) expression and localization of proteins encoding for potassium and chloride ion channels. A flow cytometric approach was used for simultaneous assessment of cell volume and viability of propidium iodide stained sperm in anisotonic media. Osmotic tolerance was found to be decreased after cryopreservation, especially in the 120 to 60 mOsm/kg osmotic range. The critical osmolality at which half of the sperm population survived increased from 55 to 89 mOsm/kg. The osmotic cell volume response for viable sperm was similar before and after cryopreservation, with an osmotic inactive volume of about 70%. The intracellular pH, determined by recording changes in carboxyfluorescein fluorescence of sperm in media with different pH before and after addition of digitonin, decreased from 6.28 in diluted sperm to 6.16 after cryopreservation. The intracellular potassium concentration, determined using the potassium ionophore nigericin and incubation in media with various potassium concentrations, increased from 154 mM to 183 mM before and after cryopreservation, respectively. The levels of the chloride and potassium ion channel proteins chloride channel 3 protein (CLC-3) and two pore domain potassium channel 2 protein (TASK-2), as detected using Western blot analysis, were not affected by cryopreservation. Immunolocalization studies showed that CLC-3 is present in the acrosome and midpiece as well as in the upper and lower tail. In conclusion, cryopreserved sperm exhibit reduced tolerance to hypotonic stress, a decreased intracellular pH, and increased intracellular potassium level.

Effect of vitrifying solutions, ethylene glycol and 1, 2-propandiol, on intracellular K+ and lactate dehydrogenase (LDH) of hepatocytes in primary culture

Shortage of donor liver tissue for the isolation of hepatocytes requires the development of improved cryopreservation techniques for long-term storage of these cells for more efficient use. The aim of this study is to compare two cryoprotectants for preserving living cells in culture by vitrification. The viability of hepatocytes after cryopreservation was determined by the intracellular concentration of potassium (K+) and lactate dehydrogenase (LDH). Cryomicroscopy was used to check the state of vitrification and morphology of these cells subjected to the process of freezing and thawing. This experiment made it possible to observe an early vitrification with 40% of Ethylene glycol (EG) or 1,2-propanedio (1, 2-PD) to reach the final vitrified state with 60%. The treatment of these cells in culture with solutions containing EG or 1, 2-PD revealed a failure in functional properties of the plasma membrane. Thus, a decrease of intracellular concentration of LDH and K+ was noticeable. This decrease was more significant when it came with 60% EG or 1, 2 - PD. The use of a cryopreservation solution additionally containing NaCl, KCl, Ca2+ and sucrose and the treatment of hepatocytes in culture at +4°C greatly minimized the functional impairment of the plasma membrane. These results suggest that the osmo-diffusional mechanism is at the basis of membrane damage letting out LDH and K+ into the extracellular compartment. However, this mechanism can be controlled by a selection of treatment solutionparameters such as temperature, osmolarity and electrolytes concentration while taking the cryoprotectants coefficient of permeability into consideration. Key words: Hepatocyte, cryopreservation, cryoprotectant, vitrified state, vitrification.

Evaluation of the Cell Viability of Human Wharton's Jelly Stem Cells for Use in Cell Therapy

Human umbilical cord Wharton's jelly stem cells (HWJSCs) are gaining attention as a possible clinical source of mesenchymal stem cells for cell therapy and tissue engineering due to their high accessibility, expansion potential, and plasticity. We employed a combination of highly sensitive techniques to determine the average cell viability levels and proliferation capabilities of 10 consecutive cell passages of cultured HWJSCs and then used RNA microarrays to identify genes associated with changes in cell viability levels. We found an initial decrease in cell viability from the first to the third cell passage followed by an increase until the sixth passage and a final decrease from the sixth to tenth cell passages. The highest cell viability levels corresponded to the fifth and sixth passages. The intracellular ionic contents of potassium, sodium, and chlorine suggest that the lower cell viability levels at passages 2, 3, and 8-10 may be associated with apoptotic cell death. In fact, gene expression analysis revealed that the average cell viability was significantly associated with genes with a function in apoptotic cell death, especially pro-apoptotic FASTKD2, BNIP3L genes and anti-apoptotic TNFAIP8 and BCL2L2 genes. This correlation with both pro-apoptotic and anti-apoptotic genes suggests that there may be a complex live-death equilibrium in cultured HWJSCs kept in culture for multiple cell passages. In this study, the highest cell viability levels corresponded to the fifth and sixth HWJSC passages, suggesting that these passages should be preferentially employed in cell therapy or tissue engineering protocols using this cell type.

Magnesium and Vascular Changes in Hypertension

Many factors have been implicated in the pathogenesis of hypertension, including changes in intracellular concentrations of calcium, sodium, potassium, and magnesium. There is a significant inverse correlation between serum magnesium and incidence of cardiovascular diseases. Magnesium is a mineral with important functions in the body such as antiarrhythmic effect, actions in vascular tone, contractility, glucose metabolism, and insulin homeostasis. In addition, lower concentrations of magnesium are associated with oxidative stress, proinflammatory state, endothelial dysfunction, platelet aggregation, insulin resistance, and hyperglycemia. The conflicting results of studies evaluating the effects of magnesium supplements on blood pressure and other cardiovascular outcomes indicate that the action of magnesium in the vascular system is present but not yet established. Therefore, this mineral supplementation is not indicated as part of antihypertensive treatment, and further studies are needed to better clarify the role of magnesium in the prevention and treatment of cardiovascular diseases.

Effects and Mechanisms of Action of Polyene Macrolide Antibiotic Nystatin on Babesia gibsoni In Vitro

Nystatin is a membrane-active polyene macrolide antibiotic and a channel-forming ionophore. Nystatin exhibits in vitro activity against Babesia gibsoni infecting normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes. The calculated IC(50) value of nystatin against B. gibsoni infecting LK erythrocytes was 31.96 µg/ml. The anti-babesial activity of nystatin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations (HK). Moreover, when the parasites were harbored in canine HK erythrocytes, which contained high potassium and low sodium concentrations as a result of high Na-K-ATPase activity, the in vitro anti-babesial activities of nystatin also disappeared, apparently due to protection by HK erythrocytes. This suggested that nystatin could show in vitro anti-babesial activity against B. gibsoni by its ionophorous activity, the same as other ionophores such as valinomycin. Subsequently, the effects of nystatin on the host cells were observed. Nystatin could not modify the intracellular concentrations of potassium, sodium, adenosine triphosphate, or glucose in either LK or HK erythrocytes, although it caused weak hemolysis in HK erythrocytes. In addition, nystatin did not affect the survival of canine peripheral polymorphonuclear leukocytes. In conclusion, nystatin destroyed B. gibsoni by ionophorous activity but did not affect either canine erythrocytes or leukocytes in vitro.

Secretion of IL-1β triggered by dynasore in murine peritoneal macrophages

The interaction of lipopolysaccharide-primed murine peritoneal macrophages with ivermectin, an antiparasite drug which potentiates P2X(4) receptors and dynasore which inhibits the GTPase activity of dynamin, a protein contributing to the internalization of plasma membrane proteins, was tested. Murine peritoneal macrophages express P2X(4) receptors which are mostly intracellular. In cells from P2X(7)-knockout mice (KO mice), 10 µm adenosine triphosphate (ATP) provoked a transient increase of the intracellular concentration of calcium. Ivermectin had no effect by itself but potentiated the increase of the intracellular concentration of calcium by ATP. The combination of ATP plus ivermectin also decreased the intracellular concentration of potassium and promoted the secretion of IL-1β. Concentrations of dynasore above 50 µm affected the integrity of mitochondria (MTT test) and of the plasma membrane (release of lactate dehydrogenase, LDH). At a 10 µm concentration, dynasore had no effect on the responses to ATP and on the internalization of P2X(4) receptors. By itself dynasore promoted the release of potassium and the secretion of IL-1β after activation of caspase-1. In conclusion, our results confirm that ivermectin potentiates the responses coupled to P2X(4) receptors probably by interaction with an allosteric site. We also show that this potentiation triggers the release of IL-1β by macrophages. As opposed to ivermectin, dynasore has no effect on P2X(4) receptors. This drug triggers a potassium efflux via a mechanism which does not involve purinergic receptors and generates, in consequence, the activation of caspase-1 and the secretion of IL-1β.

MSU, the adjuvans of dying cells activates the NALP3 inflammasome by sodium overload

Background and objectivesMonosodium urate (MSU) is one of the endogenous danger molecules generated during apoptosis. Precipitated MSU crystals in tissues and joints induce an inflammatory process with interleukin 1 β...

NKCC1 cotransporters: keeping an ‘ion’ them

NKCC1 cotransporters: keeping an ‘ion’ them