Epileptiform activity in mouse hippocampal slices induced by moderate changes in extracellular Mg2+, Ca2+, and K.

BACKGROUND: Rodent brain slices-particularly hippocampal slices-are widely used in experimental investigations of epileptiform activity. Oxygenated artificial cerebrospinal fluid (ACSF) is used to maintain slices in vitro. Physiological or standard ACSF containing 3-3.5 mM K+, 1-2 mM Mg2+, and 1-3 mM Ca2+ generally does not induce population epileptiform activity, which can be induced by ACSF with high K+ (8-10 mM), low Mg2+, or low Ca2+ alone or in combination. While low-Mg2+ ACSF without intentionally added Mg salt but with contaminating Mg2+ (≤ 50-80 µM) from other salts can induce robust epileptiform activity in slices, it is unclear whether such epileptiform activity can be achieved using ACSF with moderately decreased Mg2+. To explore this issue, we examined the effects of moderately modified (m)ACSF with 0.8 mM Mg2+, 1.3 mM Ca2+, and 5.7 mM K+ on induction of epileptiform discharges in mouse hippocampal slices. RESULTS: Hippocampal slices were prepared from young (21-28 days old), middle-aged (13-14 months old), and aged (24-26 months old) C57/BL6 mice. Conventional thin (0.4 mm) and thick (0.6 mm) slices were obtained using a vibratome and pretreated with mACSF at 35-36 °C for 1 h prior to recordings. During perfusion with mACSF at 35-36 °C, spontaneous or self-sustained epileptiform field potentials following high-frequency stimulation were frequently recorded in slices pretreated with mACSF but not in those without the pretreatment. Seizure-like ictal discharges were more common in thick slices than in thin slices. CONCLUSIONS: Prolonged exposure to mACSF by pretreatment and subsequent perfusion can induce epileptiform field potentials in mouse hippocampal slices.

Cytotoxicity of Potassium Salts of (+)- and (-) Usnic Acid for Paramecium caudatum.

We analyzed cytotoxicity of water-soluble potassium salts of (+)- and (-) usnic acid (UA) for ciliates P. caudatum. The median lethal concentrations for (+)- and (-) enantiomers did not significantly differ and were 7.5±0.5 and 6.7±0.4, respectively. In a concentration of 8 µM, (+)-UA and (-)-UA salts increased the content of TBA-reactive products, which indicates the formation of oxidative stress under the action of high UA concentrations. In the presence of (+)-UA and (-)-UA salts in a concentration range from 2 to 8 µM, the number of food vacuoles in ciliates decreased, which attested to a decrease in phagocytosis activity. The concentrations of UA enantiomers >0.5 µM affected macronucleus morphology (shape and size). The cytotoxic activity of (+)-UA and (-)-UA salts against P. caudatum did not differ.

Sensitivity of larval and juvenile freshwater mussels (unionidae) to ammonia, chloride, copper, potassium, and selected binary chemical mixtures.

In aquatic environments, organisms such as freshwater mussels are likely exposed to complex contaminant mixtures related to industrial, agricultural, and urban activities. With growing interest in understanding the risk that chemical mixtures pose to mussels, this investigation focused on the effects of various waterborne contaminants (ammonia, chloride, copper, and potassium) and selected binary mixtures of these chemicals following a fixed-ratio design to Villosa iris glochidia and juvenile Lampsilis fasciola. In individual exposures, 48-h EC50 values were determined for V. iris glochidia exposed to ammonia chloride (7.4 [95% confidence interval (CI) 6.6-8.2] mg N/L), ammonia sulfate (8.4 [7.6-9.1] mg N/L), copper sulfate (14.2 [12.9-15.4] µg Cu2+/L), potassium chloride (12.8 [11.9-13.7] mg K+/L), potassium sulfate (10.1 [8.9-11.2] mg K+/L), and sodium chloride (480.5 [435.5-525.5] mg Cl-/L). The 7-d LC50 values for juvenile L. fasciola were determined for potassium sulfate (45.0 [18.8-71.2] mg K+/L), and sodium chloride (1738.2 [1418.6-2057.8] mg Cl-/L). In Ontario these waterborne contaminants have been reported to co-occur, with concentrations exceeding the EC10 for both life stages at some locations. Data from binary mixture exposures for V. iris glochidia (chloride-ammonia, chloride-copper, and copper-ammonia) and juvenile L. fasciola (chloride-potassium) were analyzed using a regression-based, dose-response mixture analysis modeling framework. Results from the mixture analysis were used to determine if an additive model for mixture toxicity [concentration addition (CA) or independent action (IA)] best described the toxicity of each mixture and if deviation towards dose-ratio (DR) or dose-level (DL) synergism/antagonism (S/A) occurred. For all glochidia binary mixture exposures, CA was the best fit model with DL deviation reported for the chloride-copper mixture and DR deviation reported for the copper-ammonia mixture. Using the model deviation ratio (MDR), the observed toxicity in all three glochidia mixture exposures were adequately described by both CA (mean = 0.71) and IA (mean = 0.97) whereas the juvenile mixture exposure was only adequately described by CA (mean = 0.64; IA mean = 0.05).

Potassium usnate toxicity against embryonic stages of the snail Biomphalaria glabrata and Schistosoma mansoni cercariae.

The snail Biomphalaria glabrata is the most important vector for Schistosoma mansoni. Control of this vector to prevent the spread of schistosomiasis is currently performed with the application of a niclosamide molluscicide, which is highly toxic to the environment. Screening of substances that show embryotoxic molluscicidal potential as well as have detrimental effects on cercariae is very relevant for the control of schistosomiasis, as the efficacy of prevention of the disease is increased if it acts as a molluscicide as well as on the cercariae of S. mansoni. The aim of this work was to evaluate the effect of potassium usnate derived from usnic acid on different stages of embryonic development of B. glabrata and on S. mansoni cercariae. After 24 h of exposure, potassium usnate showed embryotoxic activity across all embryonic stages. The values obtained from the LC50 for the embryonic stages were the following: blastula 5.22 µg/mL, gastrula 3.21 µg/mL, trochophore 3.58 µg/mL, veliger 2.79, and hippo stage 2.52 µg/mL. Against S. mansoni cercariae, it had LC90 and 100% mortality at concentrations of 2.5 and 5 µg/mL in 2 h of exposure. In conclusion, this is the first report of potassium usnate toxicity on the embryonic stages of B. glabrata and cercariae of S. mansoni, and this study shows the potassium usnate as a promising agent for the control of mansoni schistosomiasis.

Saccular otoconia as a cause of Ménière's disease: hypothesis based on two theories.

BACKGROUND: The cause of Ménière's disease remains enigmatic after 156 years. Schuknecht's rupture and potassium intoxication theory of attacks was based on histological studies. OBJECTIVES: This paper aimed to: present the most contemporary evidence indicating that ruptures do not usually occur, and discuss the possibility that detached saccular otoconia are the main cause of Ménière's disease; and to establish an unequivocal definition of the age of Ménière's disease onset. METHOD: The paper reviews the electrophysiological basis of the Gibson-Arenberg drainage theory used to explain vertigo attacks. The current, limited knowledge of the likely fate of detached saccular otoconia is discussed. RESULTS: Electrophysiological studies during attacks do not support endolymph ruptures, but rather endolymph flowing in one direction and then in the opposite direction. Age of onset for Ménière's disease parallels that for benign paroxysmal positional vertigo. CONCLUSION: The similarity of age of onset spectrum for Ménière's disease and benign paroxysmal positional vertigo raises the possibility that the two conditions have the same fundamental cause.

Toxicologic evaluation of potassium polyaspartate (A-5D K/SD): Genotoxicity and subchronic toxicity.

Potassium polyaspartate (A-5D K/SD) is proposed for use as a stabiliser in wine, with a maximum use level of 300 mg/L and typical levels in the range of 100-200 mg/L. Potassium polyaspartate (A-5D K/SD) tested negative in a bacterial reverse mutation assay performed in accordance with OECD TG 471 and in an in vitro mammalian cell micronucleus test performed in accordance with OECD TG 487. From a 90-day oral toxicity study in male and female Wistar rats performed in accordance with OECD TG 408, a no observed adverse effect level (NOAEL) was set at 1000 mg/kg bw per day, the highest dose tested. In its opinion adopted on 9 March 2016, the EFSA-ANS Panel (European Food Safety Authority - Panel on Food Additives and Nutrient Sources added to Food), considering these data, concluded that "there was no safety concern from the proposed use and use levels of potassium polyaspartate (A-5D K/SD)".

Acute and chronic toxicity of sodium sulfate to four freshwater organisms in water-only exposures.

The acute and chronic toxicity of sulfate (tested as sodium sulfate) was determined in diluted well water (hardness of 100 mg/L and pH 8.2) with a cladoceran (Ceriodaphnia dubia; 2-d and 7-d exposures), a midge (Chironomus dilutus; 4-d and 41-d exposures), a unionid mussel (pink mucket, Lampsilis abrupta; 4-d and 28-d exposures), and a fish (fathead minnow, Pimephales promelas; 4-d and 34-d exposures). Among the 4 species, the cladoceran and mussel were acutely more sensitive to sulfate than the midge and fathead minnow, whereas the fathead minnow was chronically more sensitive than the other 3 species. Acute-to-chronic ratios ranged from 2.34 to 5.68 for the 3 invertebrates but were as high as 12.69 for the fish. The fathead minnow was highly sensitive to sulfate during the transitional period from embryo development to hatching in the diluted well water, and thus, additional short-term (7- to 14-d) sulfate toxicity tests were conducted starting with embryonic fathead minnow in test waters with different ionic compositions at a water hardness of 100 mg/L. Increasing chloride in test water from 10 mg Cl/L to 25 mg Cl/L did not influence sulfate toxicity to the fish, whereas increasing potassium in test water from 1 mg K/L to 3 mg K/L substantially reduced the toxicity of sulfate. The results indicate that both acute and chronic sulfate toxicity data, and the influence of potassium on sulfate toxicity to fish embryos, need to be considered when environmental guidance values for sulfate are developed or refined.

Effects of environmentally relevant mixtures of major ions on a freshwater mussel.

The Clinch and Powell Rivers (Virginia, USA) support diverse mussel assemblages. Extensive coal mining occurs in both watersheds. In large reaches of both rivers, major ion concentrations are elevated and mussels have been extirpated or are declining. We conducted a laboratory study to assess major ion effects on growth and survival of juvenile Villosa iris. Mussels were exposed to pond water and diluted pond water with environmentally relevant major ion mixtures for 55 days. Two treatments were tested to mimic low-flow concentrations of Ca(2+), Mg(2+), [Formula: see text] , [Formula: see text] , K(+) and Cl(-) in the Clinch and Powell Rivers, total ion concentrations of 419 mg/L and 942 mg/L, respectively. Mussel survival (>90%) and growth in the two treatments showed little variation, and were not significantly different than in diluted pond water (control). Results suggest that major ion chronic toxicity is not the primary cause for mussel declines in the Clinch and Powell Rivers.

Cesium Toxicity Alters MicroRNA Processing and AGO1 Expressions in Arabidopsis thaliana.

MicroRNAs (miRNAs) are short RNA fragments that play important roles in controlled gene silencing, thus regulating many biological processes in plants. Recent studies have indicated that plants modulate miRNAs to sustain their survival in response to a variety of environmental stimuli, such as biotic stresses, cold, drought, nutritional starvation, and toxic heavy metals. Cesium and radio-cesium contaminations have arisen as serious problems that both impede plant growth and enter the food chain through contaminated plants. Many studies have been performed to define plant responses against cesium intoxication. However, the complete profile of miRNAs in plants during cesium intoxication has not been established. Here we show the differential expression of the miRNAs that are mostly down-regulated during cesium intoxication. Furthermore, we found that cesium toxicity disrupts both the processing of pri-miRNAs and AGONOUTE 1 (AGO1)-mediated gene silencing. AGO 1 seems to be especially destabilized by cesium toxicity, possibly through a proteolytic regulatory pathway. Our study presents a comprehensive profile of cesium-responsive miRNAs, which is distinct from that of potassium, and suggests two possible mechanisms underlying the cesium toxicity on miRNA metabolism.

Avaliação da contaminação por bromato em pães do tipo francês / Analysis on the occurrence of bromate contamination in French type bread

O setor de panificação no Brasil tem realizado esforços para acompanhar as tendências de um mercadoexigente e competitivo, e uma das alternativas tem sido os agentes oxidantes. O bromato de potássio(KBrO3) é um agente oxidante que fortalece as cadeias de glúten. Apesar de benefícios tecnológicose econômicos, sua toxicidade como aditivo alimentar tem sido demonstrada. No Brasil o emprego doKBrO3 é proibido em qualquer quantidade nas farinhas e nos produtos de panificação. Foram investigadasamostras de pão Francês provenientes de 25 pontos comerciais, que foram analisadas em triplicatas,totalizando-se 75 ensaios. A determinação de bromatos foi realizada em duas etapas: primeiramente pelaprova de triagem utilizando-se o método analítico qualitativo, para identificar amostras positivas paraagentes oxidantes e, posteriormente, a determinação de BrO3- usando o reativo fucsina-bissulfito. Em todasas amostras foram detectados agentes oxidantes. Na análise confirmatória de bromato, foi identificadaa presença deste aditivo em amostras de seis estabelecimentos dos 25 analisados, correspondendo àfrequência de 24 % nas amostras examinadas. Este estudo mostra que apesar da proibição do emprego deKBrO3 em produtos de panificação, há ainda o uso deste componente pelos estabelecimentos comerciais,e isto representa risco à saúde da população...

Biocompatible KMnF3 nanoparticular contrast agent with proper plasma retention time for in vivo magnetic resonance imaging.

Nanoparticular MRI contrast agents are rapidly becoming suitable for use in clinical diagnosis. An ideal nanoparticular contrast agent should be endowed with high relaxivity, biocompatibility, proper plasma retention time, and tissue-specific or tumor-targeting imaging. Herein we introduce PEGylated KMnF3 nanoparticles as a new type of T1 contrast agent. Studies showed that the nanoparticular contrast agent revealed high bio-stability with bovine serum albumin in PBS buffer solution, and presented excellent biocompatibility (low cytotoxicity, undetectable hemolysis and hemagglutination). Meanwhile the new contrast agent possessed proper plasma retention time (circulation half-life t1/2 is approximately 2 h) in the body of the administrated mice. It can be delivered into brain vessels and maintained there for hours, and is mostly cleared from the body within 48 h, as demonstrated by time-resolved MRI and Mn-biodistribution analysis. Those distinguishing features make it suitable to obtain contrast-enhanced brain magnetic resonance angiography. Moreover, through the process of passive targeting delivery, the T1 contrast agent clearly illuminates a brain tumor (glioma) with high contrast image and defined shape. This study demonstrates that PEGylated KMnF3 nanoparticles represent a promising biocompatible vascular contrast agent for magnetic resonance angiography and can potentially be further developed into an active targeted tumor MRI contrast agent.

An ecotoxicological evaluation of aged bottom ash for use in constructions.

Municipal and Industrial Solid Waste Incineration (MISWI) bottom ash is mainly deposited in landfills, but natural resources and energy could be saved if these ash materials would be used in geotechnical constructions. To enable such usage, knowledge is needed on their potential environmental impact. The aim of this study was to evaluate the ecotoxicity of leachates from MISWI bottom ash, aged for five years, in an environmental relevant way using a sequential batch leaching method at the Liquid/Solid-ratio interval 1-3, and to test the leachates in a (sub)chronic ecotoxicity test. Also, the leachates were characterized chemically and with the technique of diffusive gradients in thin films (DGTs). By comparing established ecotoxicity data for each element with chemically analysed and labile concentrations in the leachates, potentially problematic elements were identified by calculating Hazard Quotients (HQ). Overall, our results show that the ecotoxicity was in general low and decreased with increased leaching. A strong correspondence between calculated HQs and observed toxicity over the full L/S range was observed for K. However, K will likely not be problematic from a long-term environmental perspective when using the ash, since it is a naturally occurring essential macro element which is not classified as ecotoxic in the chemical legislation. Although Cu was measured in total concentrations close to where a toxic response is expected, even at L/S 3, the DGT-analysis showed that less than 50% was present in a labile fraction, indicating that Cu is complexed by organic ligands which reduce its bioavailability.

Role of silicon in alleviating salt-induced toxicity in white clover.

To understand the role of silicon (Si) in alleviating sodium (Na) toxicity in Trifolium repens L. (white clover), the changes of biochemical and physiological parameters were investigated in four-week-old white clover seedlings exposed to 0 or 120 mM NaCl with or without 1.5 mM Si for 7 days. Results showed that added Si alone did not have any effects on the growth and Na⁺, K⁺ accumulations in white clover plants compared to the control (no added Si and NaCl). However, in the presence of NaCl, additional Si significantly enhanced the selective transport capacity for K⁺ over Na⁺ that contributed to reduced Na⁺ uptake and increased K⁺ uptake by roots, thereby improving its growth and K⁺/Na⁺ homeostasis in white clover. This study would provide a way for improving salt tolerance in important legume white clover forage.

Ion-selective microelectrode measurements of Tl⁺ and K⁺ transport by the gut and associated epithelia in Chironomus riparius.

Thallium (Tl) is a non-essential metal that is mobilized through industrial processes, subsequently entering aquatic environments where it can exert toxic effects. Although the aquatic larvae of the midge, Chironomus riparius, are exceptionally tolerant toward many waterborne non-essential metals, few studies have looked at the cellular mechanism of this tolerance. Tl⁺ and K⁺ share the same charge and have similar ionic radii, resulting in competition between these ions for K⁺ transporters. Using a recently developed Tl⁺-selective microelectrode in conjunction with the scanning ion selective electrode technique (SIET) and a two-microelectrode holder, measurements of K⁺ and Tl⁺ fluxes were made along the anal papillae and also along the isolated gut tract and Malpighian tubules (MTs) of C. riparius larvae. The MTs are a site of Tl⁺ secretion (i.e. from hemolymph into the tubule lumen). The major K⁺ transporting regions of the gut were the caecae, anterior midgut (AMG) and posterior midgut (PMG) in Tl⁺-naïve larvae, and Tl⁺ was also transported in the same direction at these locations. When the bathing saline concentration of Tl⁺ was increased to 50 µmol l⁻¹, K⁺ transport was inhibited at the AMG and PMG. Larvae exposed to 300 µmol l⁻¹ waterborne Tl⁺ for 48 h prior to ion flux measurements absorbed Tl⁺ (lumen to hemolymph) across the caecae, AMG and PMG. K⁺ secretion at the caecae was unaffected by Tl⁺ exposure, consistent with separate pathways for Tl⁺ and K⁺ transport across the caecae. By contrast, K⁺ flux at the AMG and PMG of Tl⁺-exposed larvae was impaired, suggesting that interference of Tl⁺ on K⁺ transport across these tissues may contribute to Tl⁺ toxicity.

Heterologous expression of a putative K+/H+ antiporter of S. coelicolor A3(2) enhances K+, acidic-pH shock tolerances, and geldanamycin secretion.

Heterologous expression of a putative K+/H+ antiporter of Streptomyces coelicolor A3(2) (designated as sha4) in E. coli and Streptomyces hygroscopicus JCM4427 showed enhanced tolerance to K+ stress, acidic-pH shock, and/or geldanamycin production under K+ stress. In a series of K+ extrusion experiments with sha4-carrying E. coli deficient in the K+/H+ antiporter, a restoration of impaired K+ extrusion activity was observed. Based on this, it was concluded that sha4 was a true K+/H+ antiporter. In different sets of experiments, the sha4-carrying E. coli showed significantly improved tolerances to K+ stresses and acidic-pH shock, whereas sha4-carrying S. hygroscopicus showed an improvement in K+ stress tolerance only. The sha4-carrying S. hygroscopicus showed much higher geldanamycin productivity than the control under K+ stress condition. In another set of experiments with a production medium, the secretion of geldanamycin was also significantly enhanced by the expression of sha4.

Relationship between birth weight and exposure to airborne fine particulate potassium and titanium during gestation.

Airborne particles are linked to numerous health impacts, including adverse pregnancy outcomes. Most studies of particles examined total mass, although the chemical structure of particles varies widely. We investigated whether mother's exposure to potassium (K) and titanium (Ti) components of airborne fine particulate matter (PM(2.5)) during pregnancy was associated with birth weight or risk of low birth weight (<2500 g) for term infants. The study population was 76,788 infants born in four counties in Connecticut and Massachusetts, US, for August 2000-February 2004. Both K and Ti were associated with birth weight. An interquartile range (IQR) increase K was associated with an 8.75% (95% confidence interval (CI): 1.24-16.8%) increase in risk of low birth weight. An IQR increase in Ti was associated with a 12.1% (95% CI: 3.55-21.4%) increase in risk of low birth weight, with an estimate of 6.41% (95% CI: -5.80-20.2%) for males and 16.4% (95% CI: 5.13-28.9%) for females. Results were robust to sensitivity analysis of first births only, but not adjustment by co-pollutants. Disentangling the effects of various chemical components is challenging because of the covariance among some components due to similar sources. Central effect estimates for infants of African-American mothers were higher than those of white mothers, although the confidence intervals overlapped. Our results indicate that exposure to airborne potassium and titanium during pregnancy is associated with lower birth weight. Associations may relate to chemical components of sources producing K and Ti.

Toxicity of major cations and anions (Na+, K+, Ca2+, Cl-, and SO4(2-)) to a macrophyte and an alga.

In many freshwater systems around the world, the concentrations of major ions (Na(+), K(+), Ca(2+), Mg(2+), Cl(-), HCO(3)(-), CO(3)(2-), and SO(4)(2-)) are exhibiting increasing trends, approaching the concentrations historically found mainly in estuaries. The objectives of the present study were to determine at what concentrations these salts are toxic to an aquatic plant and a green alga, to investigate two potential mechanisms of toxicity, and to determine the usefulness of conductivity as an indicator of salt toxicity. In a series of laboratory trials, Lemna minor and Pseudokirchneriella subcapitata were exposed to a range of concentrations of five different salts. Conductivity levels that caused 10 or 50% reductions in growth-related traits (EC10 and EC50, respectively) were determined, using conductivity of the test solutions as the independent variable. The EC10 values ranged from 0.44 to 2.67 mS/cm for P. subcapitata and from 1.3 to >19 mS/cm for L. minor. The EC50 values ranged from 1.7 to 5.8 mS/cm for P. subcapitata and from 4.2 to >27 mS/cm for L. minor. For both species the EC values varied dramatically among the salts. Pseudokirchneriella subcapitata was most sensitive to KCl and NaCl, whereas L. minor was most sensitive to Na(2)SO(4). The mechanism of toxicity does not appear to be related to production of reactive oxygen species, nor to reduction in chlorophyll concentrations. Because toxicity was strongly influenced by salt composition, regulation and management of specific ions may be preferable to conductivity.

Harmonic nanocrystals for biolabeling: a survey of optical properties and biocompatibility.

Nonlinear optical nanocrystals have been recently introduced as a promising alternative to fluorescent probes for multiphoton microscopy. We present for the first time a complete survey of the properties of five nanomaterials (KNbO(3), LiNbO(3), BaTiO(3), KTP, and ZnO), describing their preparation and stabilization and providing quantitative estimations of their nonlinear optical response. In the light of their prospective use as biological and clinical markers, we assess their biocompatibility on human healthy and cancerous cell lines. Finally, we demonstrate the great potential for cell imaging of these inherently nonlinear probes in terms of optical contrast, wavelength flexibility, and signal photostability.

Predicting effects of cations on copper toxicity to lettuce (Lactuca sativa) by the biotic ligand model.

A biotic ligand model (BLM) was developed to estimate Cu toxicity to lettuce (Lactuca sativa) in terms of root elongation after 4 d of exposure. Effects of Na(+), K(+), Ca(2+), and Mg(2+) on Cu toxicity were examined. The addition of these cations resulted in a 50-fold difference in the copper median effective activity (EC50 cu2+). However, these variations could not be interpreted entirely as a function of the concentrations of these cations alone. In particular, only the relationship between EC50 cu2+ and the activity of protons was found to be significant in the whole range of pH examined from 5.0 to 7.0. The addition of K(+), Na(+), Ca(2+), and Mg(2+) at concentrations up to 20 mM resulted in a 16-fold difference in EC50 cu2+ values. This difference was significant, as indicated by non-overlapping standard deviations of the negative logarithm of EC50 cu2+ pEC50 cu2+) obtained with (7.37 ± 0.22) and without (6.76 ± 0.22) additions of K(+), Na(+), Ca(2+), and Mg(2+). The variations were not statistically significantly related to concentrations of these cations; therefore, only protons can be integrated in the BLM predicting Cu toxicity to lettuce L. sativa with the important parameters: log K(HBL) =6.27, log K(CuBL) =7.40, and [formula in text]. The lack of significant relationships between EC50 cu2+ and concentrations of the cations was not in line with the main assumption of the BLM about the competition between cations for binding sites.