Participation of vacuoles in regulation of levels of K+, Mg2+ and orthophosphate ions in cytoplasm of the yeast Saccharomyces carlsbergensis

Intracellular distributions of K⁺, Mg²⁺ and orthophosphate under various conditions of cultivation or incubation of the yeast Saccharomyces carlsbergensis were studied by differential extraction of ion pools. The decisive role of vacuolar compartmentation of ions in regulation of K⁺, Mg²⁺ and orthophosphate levels in the yeast cytoplasm was shown. The content of intracellular K⁺ and Mg²⁺ in yeast increased or decreased primarily depending on the increase or decrease in the vacuolar ion pool. The levels of K⁺ and Mg²⁺ in the cytoplasm were practically unchanged. Vacuoles were involved in regulation of Mn²⁺ concentration in the cytoplasm of the yeast S. carlsbergensis accumulating this ion in the presence of glucose. Alongside the vacuolar compartmentation, the chemical compartmentation, i. e. formation of bound Mg²⁺, Mn²⁺ and K⁺ was, evidently, also involved in the control of ion levels in the cytoplasm. The orthophosphate level in the yeast cytoplasm was regulated by its accumulation in vacuoles and biosynthesis of inorganic polyphosphates in these organelles. The biosynthesis of low-molecular weight polyphosphates occurred parallel to the accumulation of Mg²⁺ or Mn²⁺ in vacuoles, thus confirming the availability of the other mechanism for the transport of these ions through the tonoplast differing from the transport mechanism through the plasmalemma.     

Fluxes of Ca2+, Sr2+ and Mg2+ in synaptosomes.

Synaptosomes isolated from sheep brain cortex accumulate Ca²⁺, Sr²⁺ and Mg²⁺ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca²⁺, 85 nmol of Mg²⁺ and 80 nmol of Sr²⁺. The loss of Ca²⁺ or Sr²⁺ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na⁺> K⁺ > Li⁺> choline, whereas for the loss of Mg²⁺ this order is different: K⁺ > Na⁺ > Li ～ choline. The efflux of Ca²⁺ or Sr²⁺ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg²⁺ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca²⁺ for Na⁺ in synaptosomes operates similarly for Sr²⁺, but not for Mg²⁺.     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

Probing the coordination properties of glutathione with transition metal ions (Cr2+,Mn2+,Fe2+,Co2+, Ni2+,Cu2+,Zn2+,Cd2+,Hg2+) by density functional theory

Complexes formed by reduced glutathione (GSH) with metal cations (Cr²⁺, Mn²⁺,Fe²⁺,Co²⁺,Ni²⁺,Cu²⁺,Zn²⁺,Cd²⁺,Hg²⁺) were systematically investigated by the density functional theory (DFT). The results showed that the interactions of the metal cations with GSH resulted in nine different stable complexes and many factors had an effect on the binding energy. Generally, for the same period of metal ions, the binding energies ranked in the order of Cu²⁺>Ni²⁺>Co²⁺>Fe²⁺>Cr²⁺>Zn²⁺>Mn²⁺; and for the same group of metal ions, the general trend of binding energies was Zn²⁺>Hg²⁺>Cd²⁺. Moreover, the amounts of charge transferred from S or N to transition metal cations are greater than that of O atoms. For Fe²⁺,Co²⁺,Ni²⁺,Cu²⁺,Zn²⁺,Cd²⁺ and Hg²⁺ complexes, the values of the Wiberg bond indices (WBIs) of M-S (M denotes metal cations) were larger than that of M-N and M-O; for Cr²⁺ complexes, most of the WBIs of M-O in complexes were higher than that of M-S and M-N. Furthermore, the changes in the electron configuration of the metal cations before and after chelate reaction revealed that Cu²⁺, Ni²⁺,Co²⁺ and Hg²⁺ had obvious tendencies to be reduced to Cu⁺,Ni⁺,Co⁺ and Hg⁺ during the coordination process.     

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture

We have addressed the possibility that Ca²⁺, Mg²⁺ and K⁺ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca²⁺, Mg²⁺ or K⁺ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K⁺ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg²⁺ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca²⁺ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg²⁺ ion deprivation and apoptosis inducing effects of Ca²⁺ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K⁺ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca²⁺ Mg²⁺ or K⁺ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.     

Super-high-affinity binding site for [3H]diazepam in the presence of Co2+, Ni2+, Cu2+, OR Zn2+

Chloride salts of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, and Fe³⁺ had no effect on [³H]diazepam binding. Chloride salts of Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ increased [³H]diazepam binding by 34 to 68% in a concentration-dependent fashion. Since these divalent cations potentiated the GABA-enhanced [³H]diazepam binding and the effect of each divalent cation was nearly additive with GABA, these cations probably act at a site different from the GABA recognition site in the benzodiazepine-receptor complex. Scatchard plots of [³H]diazepam binding without an effective divalent cation showed a single class of binding, with a Kd value of 5.3 mM. In the presence of 1 mM Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺, two distinct binding sites were evident with apparent Kd values of 1.0 nM and 5.7 nM. The higher-affinity binding was not detected in the absence of an effective divalent cation and is probably a novel, super-high-affinity binding site.     

Perturbation of Hydrogen Bonds in the Adenine…Thymine Base Pair by Na+, Mg2+, Ca2+ and NH4 + Cations

Abstract

Perturbation of the hydrogen bonds in the adenine…thymine base pair by Na⁺, Mg²⁺, Ca²⁺ and NH₄ ⁺ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine…thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at 06 leads to destabilization of the adenine…thy mine pair; divalent cations (Mg²⁺, Ca²⁺) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+ and Zn2+) and water coordination on the structure and properties of l-histidine and zwitterionic l-histidine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. The effect of metal ions and water on the structure of l-histidine is examined. The effect of metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺) and water on structures of His·M(H₂O)m, m = 0.1 complexes have been determined theoretically employing density functional theories using extended basis sets. Of the five stable complexes investigated the relative stability of the gas-phase complexes computed with DFT methods (with one exception of K⁺ systems) suggest metallic complexes of the neutral l-histidine to be the most stable species. The calculations of monohydrated systems show that even one water molecule has a profound effect on the relative stability of individual complexes. Proton dissociation enthalpies and Gibbs energies of l-histidine in the presence of the metal cations Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ were also computed. Its gas-phase acidity considerably increases upon chelation. Of the Lewis acids investigated, the strongest affinity to l-histidine is exhibited by the Cu²⁺ cation. The computed Gibbs energies ΔG are negative, span a rather broad energy interval (from ?130 to ?1,300 kJ/mol), and upon hydration are appreciably lowered.     

Competitive membrane adsorption of Na+, K+, and Ca2+ in smooth muscle cells

Summary A theory for Na⁺, K⁺ and Ca²⁺ competitive adsorption to a charged membrane is used to explain a number of experimental observations in smooth muscle. Adsorption is described by Langmuir isotherms for mono- and divalent cations which in turn are coupled in a self-consistent way to the bulk solution through the diffuse double layer theory and the Boltzman equations. We found that the dissociation constants for binding of Na⁺, K⁺ and Ca²⁺ in guinea pig taenia coli areca. 0.009, 1.0, and 4×10^–8 m, respectively. Furthermore, the effect of a Ca²⁺ pump that maintains free surface Ca²⁺ concentration constant is investigated. A decrease in intracellular Na⁺ content results in an increased Ca²⁺ uptake; part of this uptake is due to an increase in surface-bound Ca²⁺ in an intracellular compartment which is in contact with the myofilaments. Variations in the amount of charge available to bind Ca²⁺ and the surface charge density are studied and their effect interpreted in terms of different pharmacological agents.     

Cation (K+, Mg2+, Ca2+) exchange in Pb2+ accumulation by Saccharomyces cerevisiae

The relationship between Pb²⁺ accumulation and cation (K⁺, Mg²⁺, Ca²⁺) release in Saccharomyces cerevisiae was extensively investigated. As Pb²⁺ accumulation proceeded, the release of cellular metal ions such as K⁺, Mg²⁺ and Ca²⁺ was concomitantly released within 24 h, thereafter Pb²⁺ penetrated into the inner cellular parts and consequently plasmolysis of the cell was observed by TEM analysis. Pb²⁺ accumulation process in S. cerevisiae after 24 h was metabolism-independent because of the absence of cell viability. As the cell storage time was prolonged, the released amount of K⁺ was markedly increased, while the amount of accumulated Pb²⁺ was nearly constant regardless of cell storage time and the time required to reach an equilibrium state was shortened. The autoclaved cells had less Pb²⁺ accumulation capacity than the untreated cells, and the amounts of released K⁺ and Mg²⁺ were very low due to the denaturation of cell surface and cell membrane.     

Mg2+, K+, and the Ribosome

Mg²⁺ and K⁺ are the prevalent di- and monovalent cations inside the cells in all three domains, playing a dominant role in structure and function of biological macromolecules. Ribosomes bind a substantial fraction of total Mg²⁺ and K⁺ cations. In this issue of the Journal of Bacteriology, Akanuma and coworkers (G. Akanuma et al., J. Bacteriol. 196:3820–3830, 2014, doi:10.1128/JB.01896-14) report a surprising genetic link between ribosome amounts per cell and the intracellular Mg²⁺ concentrations.     

Interactions among Ca2+, Na+ and K+ in salinity toxicity: quantitative resolution of multiple toxic and ameliorative effects

Root elongation by wheat seedlings (Triticum aestivum L. cv. Scout 66) was not inhibited by NaCl or KCl up to 130 mM in culture solutions or by high Na⁺ (2 mg g^-1 FW) or K⁺ (4 mg g^-1 FW) in the root tissue, provided that [Ca²⁺]>2 mM in the rooting medium. At [NaCl], [KCl], or [mannitol] >250 mOs, root elongation was progressively inhibited, irrespective of high [Ca²⁺]. In contrast, shoot elongation was sensitive to any diminution of water potential, and Ca²⁺ alleviated the toxicity only weakly. At solute concentrations <250 mOs, the following interactions were observed. Ca²⁺ alleviated Na⁺ and K⁺ toxicity to roots by at least three separate mechanisms. K⁺ was more toxic to roots than Na⁺, but Na⁺ was more toxic to shoots. Low levels of K⁺ relieved Na⁺ toxicity, but low levels of Na⁺ enhanced K⁺ toxicity. Tissue concentrations of Na⁺ were reduced by Ca²⁺ and K⁺ in the rooting medium, and tissue concentrations of K⁺ were enhanced by Ca²⁺ and Na⁺. Several hypotheses relating to salinity toxicity can be evaluated, at least for wheat seedlings. The osmoticant hypotheses (salinity intoxication occurs because of diminished water potential) is true for shoots at all salinity levels, but is true for roots only at high salinity. The Ca²⁺-displacement hypothesis (Na⁺ is toxic because it displaced Ca²⁺ from the cell surface) is correct, but often of minor importance. The K⁺-depletion hypothesis (Na⁺ is toxic because it causes a loss of K⁺ from plant tissues) is false. The Cl^--toxicity hypothesis (the apparent toxicity of Na⁺ is induced by associated Cl^-) is false. The results indicate that, apart from osmotic effects, high levels of Na⁺ in the rooting medium and in the tissues are not toxic unless Ca²⁺ is also deficient, a condition probably leading to inadequate compartmentation and excessive cytoplasmic accumulation. This study related growth to ion activities at plasma-membrane surfaces. These activities were computed by a Gouy-Chapman-Stern model then incorporated into non-linear growth models for growth versus toxicants and ameliorants.Key words: Calcium, potassium, salinity, sodium, toxicity      

cAMP activates hyperpolarization-activated Ca2+ channels in the pollen of Pyrus pyrifolia

Many signal-transduction processes in plant cells have been suggested to be triggered by signal-induced opening of calcium ion (Ca²⁺) channels in the plasma membrane. Cyclic nucleotides have been proposed to lead to an increase in cytosolic free Ca²⁺ in pollen. However, direct recordings of cyclic-nucleotide-induced Ca²⁺ currents in pollen have not yet been obtained. Here, we report that cyclic AMP (cAMP) activated a hyperpolarization-activated Ca²⁺ channel in the Pyrus pyrifolia pollen tube using the patch-clamp technique, which resulted in a significant increase in pollen tube protoplast cytosolic-Ca²⁺ concentration. Outside-out single channel configuration identified that cAMP directly increased the Ca²⁺ channel open-probability without affecting channel conductance. cAMP-induced currents were composed of both Ca²⁺ and K⁺. However, cGMP failed to mimic the cAMP effect. Higher cytosolic free-Ca²⁺ concentration significantly decreased the cAMP-induced currents. These results provide direct evidence for cAMP activation of hyperpolarization-activated Ca²⁺ channels in the plasma membrane of pollen tubes, which, in turn, modulate cellular responses in regulation of pollen tube growth.     

Combined actions of Pb2+, Zn2+, and Al3+ on voltage-activated calcium channel currents

Summary 1. Whole-cell patch clamp experiments were performed on rat dorsal root ganglion (DRG) neurons to investigate the actions of various combinations of Pb²⁺, Zn²⁺, and Al³⁺ on voltage-activated calcium channel currents (VACCCs).2. Each of these metals has been shown to reduce VACCCs.3. We investigated the effects of simultaneous application of two cations in the range of their IC₅₀ values. For all possible combinations (Pb²⁺/Zu²⁺, Zn²⁺/Al³⁺, Al³⁺/Pb²⁺), independent of the order of application, we found additive actions on VACCCs.4. We observed a 75% (±9%) block of the control current when two cations were applied simultaneously. This observation is consistent with both, an action of two metals at the same site as well as independent actions at different locations of the ion channel.5. The additivity of the effects should be taken into account for questions of public health and the assessment of threshold limits in cases of environmental contamination.     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.     

Boron deficiency alters cytosolic Ca2+ concentration and affects the cell wall components of pollen tubes in Malus domestica

• Boron (B) is essential for normal plant growth, including pollen tube growth. B deficiency influences various physiological and metabolic processes in plants. However, the underlying mechanism of B deficiency in pollen tube growth is not sufficiently understood. In the present research, the influence of B deficiency on apple (Malus domestica) pollen tube growth was studied and the possible regulatory mechanism evaluated.
• Apple pollen grains were cultured under different concentrations of B. Scanning ion‐selective electrode technique, fluorescence labelling and Fourier‐transform infrared (FTIR) analysis were used to detect calcium ion flux, cytosolic Ca²⁺ concentration ([Ca²⁺]cyt), actin filaments and cell wall components of pollen tubes.
• B deficiency inhibited apple pollen germination and induced retardation of tube growth. B deficiency increased extracellular Ca²⁺ influx and thus led to increased [Ca²⁺]cyt in the pollen tube tip. In addition, B deficiency modified actin filament arrangement at the pollen tube apex. B deficiency also altered the deposition of pollen tube wall components. Clear differences were not observed in the distribution patterns of cellulose and callose between control and B deficiency treated pollen tubes. However, B deficiency affected distribution patterns of pectin and arabinogalactan proteins (AGP). Clear ring‐like signals of pectins and AGP on control pollen tubes varied according to B deficiency. B deficiency further decreased acid pectins, esterified pectins and AGP content at the tip of the pollen tube, which were supported by changes in chemical composition of the tube walls.
• B appears to have an active role in pollen tube growth by affecting [Ca²⁺]cyt, actin filament assembly and pectin and AGP deposition in the pollen tube. These findings provide valuable information that enhances our current understanding of the mechanism regulating pollen tube growth.

     

Calcium snapshots in the stigma and style of medlar (Lycium barbarum L.) during pollen germination and pollen tube growth suggest active calcium oscilations

Abstract

The stigma (tip of the pistil) of medlar is wet and covered with stigmatic exudate at anthesis. The exudate contains many vesicles with abundant calcium precipitates. After deposition on the stigma, the pollen grain undergoes hydration, displaying signs of calcium ion (Ca²⁺) transfer from the exudate vesicles into the pollen grains. Calcium precipitates in the pollen cytoplasm are concentrated into small vacuoles that fuse to form large vacuoles, which provide turgor pressure to push the cytoplasm to the apical region of the growing pollen tube. Many calcium precipitates are present in the stylar transmitting tract, which displays a calcium gradient: fewer precipitates are localised in the distal (upper) transmitting tissue below the stigma, and more precipitates are present in the transmitting tract at the style base. The emporal and spatial distribution of calcium in the stigma and style of medlar suggests that it satisfies the demand for calcium in vivo and played some functional significance.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Al3+-Ca2+ interactions in aluminum rhizotoxicity

Several mineral rhizotoxicities, including those induced by Al³⁺, H⁺, and Na⁺, can be relieved by elevated Ca²⁺ in the rooting medium. This leads to the hypothesis that the toxic cations displace Ca²⁺ from transport channels or surface ligands that must be occupied by Ca²⁺ in order for root elongation to occur. In this study with wheat (Triticum aestivum L.) seedlings, we have determined, in the case of Al³⁺, that (i) Ca²⁺, Mg²⁺, and Sr²⁺ are equally ameliorative, (ii) that root elongation does not increase as Ca²⁺ replaces Mg²⁺ or Sr²⁺ in the rooting media, and (iii) that rhizotoxicity is a function solely of Al³⁺ activity at the root-cell membrane surface as computed by a Gouy-Chapman-Stern model. The rhizotoxicity was indifferent to the computed membrane-surface Ca²⁺ activity. The rhizotoxicity induced by high levels of tris(ethylenediamine)cobaltic ion (TEC³⁺), in contrast to Al³⁺, was specifically relieved by Ca²⁺ at the membrane surface. The rhizotoxicity induced by H⁺ exhibited a weak specific response to Ca²⁺ at the membrane surface. We conclude that the Ca²⁺-displacement hypothesis fails in the case of Al³⁺ rhizotoxicity and that amelioration by cations (including monovalent cations) occurs because of decreased membrane-surface negativity and the consequent decrease in the membrane-surface activity of Al³⁺. However, TEC³⁺, but not Al³⁺, may be toxic because it inhibits Ca²⁺ uptake. The nature of the specific H⁺-Ca²⁺ interaction is uncertain.Abbreviations {Al³⁺ }₀ chemical activity of Al³⁺ at the root-cell membrane surface - {Al³⁺ }_E chemical activity of Al³⁺ in the external rooting medium - E₀ electrical potential at the root-cell membrane surface - HXM²⁺ hexamethonium ion - TEC³⁺ tris(ethylenediamine)cobaltic ion