Potassium-selective Ionophore Research Articles

Impedance-based sensor for potassium ions

A conductometric sensor for potassium ions in solution is presented. Interdigitated, planar gold electrodes were coated with a potassium-selective polymer membrane composed of a poly(vinyl chloride) matrix with about 65 wt% of plasticiser and 2–5 wt% of a potassium-selective ionophore. The impedance of the membrane was measured, using the electrodes as a transducer, and related to the concentration of potassium in a sample solution in contact with the membrane. Sensitivity was optimised by varying the sensor components, and selectivity for potassium over sodium was also shown. The resulting devices are compact, miniature, robust sensors which, by means of impedance measurements, eliminate the need for a reference electrode. The sensor was tested for potassium concentration changes of 2 mM across the clinically relevant range of 2.7–18.7 mM.

The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation

The quality of mitochondria, essential organelles that produce ATP and regulate numerous metabolic pathways, must be strictly monitored to maintain cell homeostasis. The loss of mitochondrial quality control systems is acknowledged as a determinant for many types of neurodegenerative diseases including Parkinson's disease (PD). The two gene products mutated in the autosomal recessive forms of familial early-onset PD, Parkin and PINK1, have been identified as essential proteins in the clearance of damaged mitochondria via an autophagic pathway termed mitophagy. Recently, significant progress has been made in understanding how the mitochondrial serine/threonine kinase PINK1 and the E3 ligase Parkin work together through a novel stepwise cascade to identify and eliminate damaged mitochondria, a process that relies on the orchestrated crosstalk between ubiquitin/phosphorylation signaling and autophagy. In this review, we highlight our current understanding of the detailed molecular mechanisms governing Parkin-/PINK1-mediated mitophagy and the evidences connecting Parkin/PINK1 function and mitochondrial clearance in neurons.

The Mechanisms of the Release of Cytochrome c from Mitochondria Revealed by Proteomics Analysis

Mitochondrial permeability transition (PT) is the phenomenon in which the mitochondrial inner membrane becomes permeable to various solutes and ions. When PT is induced by Ca(2+), cytochrome c is released from mitochondria into the cytosol where it then triggers subsequent steps of programmed cell death, apoptosis. Thus, the proteins that regulate PT and cytochrome c release could become druggable targets for various diseases. However, the mechanisms of PT and the release of cytochrome c have not yet been revealed. We previously showed that valinomycin, a potassium selective ionophore, also caused release of cytochrome c from mitochondria without inducing PT. This result indicates that cytochrome c could be released from mitochondria with or without induction of PT. In this study, to understand the difference of effects of valinomycin and Ca(2+) on mitochondria, we examined what protein species are released from valinomycin- and Ca(2+)-treated mitochondria by LC-MS/MS. As a result, only the proteins located in the intermembrane space were found to be released from valinomycin-treated mitochondria, while those in both the intermembrane space and in the matrix were released from Ca(2+)-treated mitochondria. Furthermore, the protein releases by each reagent occurred not selectively but in a concentration-dependent manner. Based on these results, the permeabilization effects of Ca(2+) and valinomycin on the inner and outer mitochondrial membranes are discussed.

Direct binding of halide ions by valinomycin

The antibiotic valinomycin is a potassium-selective ionophore, which increases the transport of potassium ions across cell membranes and thereby causes damage to bacteria cells. Valinomycin has been extensively studied as an ionophore for cations. We report for the first time the direct binding of anions to valinomycin using electrospray ionisation mass spectrometry and 1H nuclear magnetic resonance (NMR) spectroscopy. The binding selectivity for halide ions is found to be in the order Cl− >Br− ∼F− ≫I− based on electrospray ionisation mass spectrometry experiments in methanol. 1H NMR studies in acetone-d 6 and CD3CN reveal the binding selectivity of Cl− >Br− ≫F− ∼I− . NMR studies and density functional theory (DFT) calculations support a bracelet-like structure for the binding of a chloride ion to valinomycin. Association constants of 531 ± 45 M− 1 and 57 ± 2 M− 1 were obtained via NMR titrations in acetone-d 6 for chloride and bromide ions, respectively.

Complexation of cyclic dodecadepsipeptide, cereulide with ammonium salts.

Cereulide is a principal toxin causing emetic syndrome which is produced by Bacillus cereus and has been known as potassium selective ionophore. This paper deals with its complexation with inorganic and organic ammonium ions to assign the higher structures similar to the complex with potassium ion by means of NMR and ESI-MS spectroscopy. Of particular interest, the detectable ions are not only at m/z 1191.8 as K(+) complex but also (or sometimes exclusively) at m/z 1170.8 as NH(4)(+) complex in its LC-MS analyses depending upon the conditions. This difference is due to the sample preparation and measurement condition.

Flow cytometric ion detection with plasticized poly(vinyl chloride) microspheres containing selective lonophores.

The use of flow cytometry as a rapid, high-throughput diagnostic tool for the analysis of ions is described. Monodisperse, uniform microspheres, which obey bulk optode theory and are governed by bulk extraction processes rather than surface phenomena, were prepared under mild, nonreactive conditions using a sonic stream particle casting apparatus. As an initial example demonstrating the utility of this approach, microspheres that contained a H+-selective fluorescent chromoionophore (ETH 5294), a cation-exchanger (NaTFPB), and either a highly sodium-selective (sodium ionophore X) or a potassium-selective ionophore (BME-44) were prepared. Separate solution analysis of sodium- and potassium-selective microspheres resulted in the generation of functional response curves using peak channel fluorescence intensities. The selectivity observed for both types of particles is sufficient for the clinical determination of Na+ and K+. Furthermore, sodium- and potassium-selective microspheres were analyzed in parallel using sodium sample solutions, resulting in the successful determination of sodium ion concentrations and providing important information about the selectivity of the potassium-selective sensors over sodium. This work demonstrates the potential applicability of flow cytometry as a means for developing multiplexed, rapid, high-throughput analyses for clinically relevant ions.

Opening of ATP-sensitive potassium channels causes generation of free radicals in vascular smooth muscle cells.

Recent evidence suggests that opening of mitochondrial K(ATP) channels in cardiac muscle triggers the preconditioning phenomenon through free radical production. The present study tested the effects of K(ATP) channel openers in a vascular smooth muscle cell model using the fluorescent probe MitoTracker (MTR) Red trade mark for detection of reactive oxygen species (ROS). Rat aortic smooth muscle cells (A7r5) were incubated with 1 micro M reduced MTR (non-fluorescent) and the MTR oxidation product (fluorescent) was quantified. Thirty-minute pretreatment with either diazoxide (200 micro M) or pinacidil (100 micro M), both potent mitochondrial K(ATP) channel openers, increased fluorescent intensity (FI) to 149 and 162 % of control (p < 0.05 for both), respectively, and the K(ATP) channel inhibitor 5-hydroxydecanoate (5 HD) blocked it. Valinomycin, a potassium-selective ionophore, raised FI to 156 % of control (p <: 0.05). However, 5 HD did not affect the valinomycin-induced increase in FI. Inhibition of mitochondrial electron transport (myxothiazol) or uncoupling of oxidative phosphorylation (dinitrophenol) also blocked either valinomycin- or diazoxide-induced increase in FI, and free radical scavengers prevented any diazoxide-mediated increase in fluorescence. Finally the diazoxide-induced increase in fluorescence was not blocked by the PKC inhibitor chelerythrine, but was by HMR 1883, a putative surface K(ATP) channel blocker. Thus opening of K(ATP) channels increases generation of ROS via the mitochondrial electron transport chain in vascular smooth muscle cells. Furthermore, a potassium-selective ionophore can mimic the effect of putative mitochondrial KATP channel openers. We conclude that potassium movement through KATP directly leads to ROS production by the mitochondria.

Perbrominated closo-dodecacarborane anion, 1-HCB11Br11-, as an ion exchanger in cation-selective chemical sensors.

The 2,3,4,5,6,7,8,9,10,1 1,12-undecabromocarborane anion, 1-HCB11Br11- (UBC-) has been evaluated for its suitability as an ion exchanger in solvent polymeric membrane electrodes and bulk optodes. Experiments comparing the chemical stability of the perhalogenated carborane anion to that of the best lipophilic tetraphenylborate, 3,5-[bis(trifluoromethyl)phenyl]borate (TFPB-), demonstrated that in the presence of 0.2 M acetic acid TFPB- was completely lost within 6 h, while the concentration of UBC- decreased by less than 10% in the same time period. Thin-film bulk optodes containing BME-44 as potassium-selective ionophore, ETH 5294 as chromo-ionophore, and UBC- as ionic sites exhibited a K+ response similar to analogous optodes containing TFPB-, with comparable selectivities over Na+ and Ca2+. Potentiometric measurements evaluating the selectivity behavior of UBC- in both ionophore-free and ionophore-containing electrodes were performed. Ionophore-free PVC membranes containing UBC- as ion exchanger and either DOS or NPOE as plasticizer also demonstrated selectivity similar to TFPB--containing membranes. Sodium-selective membranes containing the ionophore 4-tert-butylcalix[4]arenetetraacetic acid tetraethyl ester (sodium ionophore X) and UBC- as ionic sites showed a Nernstian response for sodium and selectivity comparable to that found in analogous electrodes containing TFPB-.

Calix[4]tubes: a new class of potassium-selective ionophore.

A new class of cryptand-like ionophore based on a bis calix[4]arene structure has been developed. These molecules proved highly selective for complexation of potassium over all group I metal cations and barium. A range of symmetric and asymmetric calix[4]tubes featuring either alkyl or phenyl substituents at the upper rim have been synthesized in exceptional yield. Alteration of the calix[4]arene upper rim environment enables fine-tuning of the rate of potassium uptake, which can be evaluated through the conformational change on binding by NMR studies. Selectivity of potassium complexation has been demonstrated using NMR and electrospray mass spectral techniques. Molecular modeling studies, derived from crystallographic data, confirm that the potassium metal cation is complexed via the axial route, passing through the calix[4]arene annulus, and provide evidence for complexation rate and selectivity.

Membrane Hyperpolarization by Sperm-Activating and -Attracting Factor Increases cAMP Level and Activates Sperm Motility in the Ascidian Ciona intestinalis

In the ascidian Ciona intestinalis (and C. savignyi), sperm-activating and -attracting factor (SAAF) is released from the egg at fertilization and stimulates both Ca2+ influx and a transient increase in cAMP level of the sperm, leading to the activation of sperm motility (M. Yoshida et al., 1994, Dev. Growth Differ. 36, 589–595). In this paper we show in C. intestinalis that valinomycin, a potassium-selective ionophore, as well as SAAF, activated sperm motility, and this activation was suppressed by extracellular high K+. Membrane potential measurements showed that both SAAF and valinomycin increase K+ permeability of sperm and induce membrane hyperpolarization, the amplitude of which depends on the external K+ concentration. The membrane potential and intracellular K+ concentration of Ciona sperm without SAAF were estimated to be about −50 mV and 560 ± 40 mM, respectively. After treatment with SAAF or valinomycin the membrane potential became almost equal to the equilibrium potential of K+ (−100 mV), and the cAMP level increased in artificial seawater. A potent voltage-dependent K+ channel blocker, MCD peptide, at the concentration of 10 μM blocked SAAF-induced hyperpolarization of the cells, increase in cAMP, and sperm motility. These results suggest that membrane hyperpolarization produced by the opening of K+ channels elevates cAMP synthesis and leads to the activation of sperm motility in Ciona.

Planar waveguide ion-selective sensors

A potassium ion-selective planar waveguide total internal reflection (TIR) mode optical sensor was prepared for flow injection application by casting thin film optode membranes onto a glass substrate. For this sensor, a potassium selective ionophore is incorporated in plasticized polyvinyl chloride film sensitized with a hydrogen selective ionophore. The sensor response to potassium ion is determined by measuring the attenuation of a HeNe laser propagated in this thin film device. The laser was coupled into and out of the sensor using grating couplers providing an inexpensive, environmentally stable sensor. The sensitivity of the waveguide based flow injection device is approximately 0.05 mM K +, while the rate of analysis is about 180 samples per hour.

Development of a fluorescent optical potassium-selective ion sensor with ratiometric response for intracellular applications

A small, fluorescent fiber-optic potassium sensor is described with a working range which covers both intracellular cytosolic and blood levels of potassium at the respective physiological pH values. The highly potassium-selective ionophore, BME-44, is retained in a plasticized poly (vinyl chloride) (PVC) film which is placed on the end of an optical fiber. Three optode configurations are given, employing different lipophilic, fluorescent pH chromoionophores (Nile Blue derivatives), demonstrating the ability to improve the detection limit and tune the dynamic range to the desired region of interest. These optodes also have excellent selectivity over sodium, calcium and magnesium. Use of fluorescence (in contrast to absorbance) provides a means to reduce the sensor size dramatically while retaining the same signal-to-noise ratio. These optodes measure the sample's potassium activity (rather than concentration) provided that the sample's pH is measured simultaneously by another sensor, such as a glass pH electrode.

Effect of surface-attached heparin on the response of potassium-selective electrodes.

Heparin (or hydrolyzed heparin) was covalently attached on the surface of derivatized cellulose triacetate membranes, which were subsequently impregnated with the potassium-selective ionophore valinomycin. The resulting ion-selective electrodes presented near-Nernstian response to potassium and had selectivity coefficients of the same order of magnitude as those of conventional poly-(vinyl chloride)-based electrodes. It was found that the heparin layer does not alter significantly the response characteristics of the electrodes. The biological activity of the immobilized heparin (or hydrolyzed heparin) was measured in terms of its inactivation of blood coagulation factor Xa. It was found that the covalently anchored hydrolyzed heparin was not biologically active, but the immobilized heparin was able to inactivate factor Xa. Therefore, by covalently attaching heparin on the surface of ion-selective electrodes, electrodes with improved blood compatibility characteristics may be prepared.

Electrical properties of individual cells isolated from adult rat ventricular myocardium.

1. Individual cells were isolated from adult rats ventricular myocardium by a collagenase digestion procedure. 2. Steady membrane potentials recorded with conventional intracellular glass micro-electrodes from cells in a modified Krebs solution containing 3 . 8 mM-KCl and 0 . 5 mM-CaCl2 were less negative than -40 mV in most cells (-25 . 3 +/- 10 . 9 mV, mean +/- S.D., 211 cells). 3. After addition of the potassium selective ionophore valinomycin (60 nM) to the bathing solution all recorded membrane potentials were more negative than -60 mV (-74 . 8 +/- 7 . 0 mV, sixty-three cells). 4. The internal concentration of potassium in the cells was determined as 120 . 8 +/- 1 . 7 mM (+/- S.E., n = 24) by flame emission spectrometry after centrifugation through silicone oil, using tritiated water and D-[1-14C] mannitol to estimate total and extracellular water in the pellet. 5. In the majority of cells in the standard solution the membrane potential recorded within a few msec of penetration was more negative than -70 mV (-78 . 4 +/- 9 . 7 mV, seventy-three cells). In sixty-six cells penetration initiated an action potential which overshot zero by 31 . 3 +/- 7 . 1 mV. This overshoot was abolished by reducing the external sodium to 0 . 1 of the normal value, and reduced or abolished by addition of tetrodotoxin (30 microM). 6. Modifications of the standard bathing solution which increased the number of cells with steady recorded membrane potentials more negative than -60 mV were: isosmotic substitution of sucrose for NaCl; replacement of NaCl and KCl by sodium isethionate and potassium methyl sulphate; addition of 5 or 10 mM-CaCl2; addition of 10 mM-MnCl2. 7. For cells in solution containing 2 . 5 or 5 . 5 mM-CaCl2, input resistances estimated from the amplitude of hyperpolarizations evoked by 200 msec current pulses were approximately 40 M omega at a resting potential close to -80 mV and became much greater as cells were depolarized. Time constants measured at the resting potential were approximately 8 msec. 8. In certain conditions, repeated spontaneous action potentials were recorded from contracting cells, and in quiescent cells evoked action potentials could be initiated by applying brief depolarizing pulses through the micro-electrode. Action potentials were coincident with contractions. 9. It is concluded that the resting potential of these isolated cells is normally more negative than -70 mV, and that the cells retain the ionic mechanisms necessary for the generation of active currents.