A study of Li+-selective permeation through lipid bilayer membranes mediated by a new ionophore (AS701)

The neutral, noncyclic, imide and ether containing ionophore AS701, has been developed as Li⁺-selective molecule, to be used potentially as an aid in the Li⁺-therapy of manic-depressive illness. The present report is a characterization of this molecule in neutral lipid bilayer membranes. This ionophore was found to the bilayers Li⁺-selective, acting as a selective carrier of monovalent cations. In addition, this molecule was found to be capable of acting as a selective carrier of monovalent anions. For both types of ions, the rate-limitting step in the process of permeation was found to be the diffusion of the carrier-ion complex through the membrane. The membrane-permeating species were found to be 2 : 1 carrier-ion complexes, carrying either a monovalent cation or a monovalent anion. The selectivity sequences among the ions studied being: Li⁺(1) > ClO₄^?(0.7) > Na⁺(0.07) > K⁺(0.016) > Rb⁺(0.0095) > Cs⁺(0.0083) > Cl^?(0.001). Mg²⁺ and SO₄^2? were found to be impermeant (under present experimental conditions). This sequence shows that the AS701 molecule has low selectivity for ions present in biological media, among those studied (i.e. Na⁺, K⁺, Mg²⁺, Cl^2? and SO₄^2?). This indicates that these ions will not interfere in the Li⁺ permeability induced by this carrier in vivo, and that the carrier will not interfere in the normal transport processes of these ions.     

Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1

The wheat root high-affinity K⁺ transporter HKT1 functions as a sodium-coupled potassium co-uptake transporter. At toxic millimolar levels of sodium (Na⁺), HKT1 mediates low-affinity Na⁺ uptake while potassium (K⁺) uptake is blocked. In roots, low-affinity Na⁺ uptake and inhibition of K⁺ uptake contribute to Na⁺ toxicity. In the present study, the selectivity among alkali cations of HKT1 expressed in Xenopus oocytes and yeast was investigated under various ionic conditions at steady state. The data show that HKT1 is highly selective for uptake of the two physiologically significant alkali cations, K⁺ and Na⁺ over Rb⁺, Cs⁺ and Li⁺. In addition, Rb⁺ and Cs⁺, and an excess of extracellular K⁺ over Na⁺, are shown to partially reduce or block HKT1-mediated K⁺-Na⁺ uptake. Furthermore, K⁺, Rb⁺ and Cs⁺ also effectively reduce outward currents mediated by HKT1, thereby causing depolarizations. In yeast, HKT1 can produce high-affinity Rb⁺ uptake at approximately 15-fold lower rates than for K⁺. Rb⁺ influx in yeast can be mediated by the ability of the yeast plasma membrane proton pump to balance the 35-fold lower HKT1 conductance for Rb⁺. A model for HKT1 activity is presented involving a high-affinity K⁺ binding site and a high-affinity Na⁺ binding site, and competitive interactions of K⁺, Na⁺ and other alkali cations for binding to these two sites. Possible implications of the presented results for physiological K⁺ and Na⁺ uptake in plants are discussed.     

Discrimination between Alkali Metal Cations by Yeast : II. Cation interactions in transport

K⁺ is a competitive inhibitor of the uptake of the other alkali metal cations by yeast. Rb⁺ is a competitive inhibitor of K⁺ uptake, but Li⁺, Na⁺, and Cs⁺ act like H⁺. At relatively low concentrations they behave as apparent noncompetitive inhibitors of K⁺ transport, but the inhibition is incomplete. At higher concentrations they inhibit the remaining K⁺ transport competitively. Ca⁺⁺ and Mg⁺⁺ in relatively low concentrations partially inhibit K⁺ transport in an apparently noncompetitive manner although their affinity for the transport site is very low. In each case, in concentrations that produce "noncompetitive" inhibition, very little of the inhibiting cation is transported into the cell. Competitive inhibition is accompanied by appreciable uptake of the inhibiting cation. The apparently noncompetitive effect of other cations is reversed by K⁺ concentrations much higher than those necessary to essentially "saturate" the transport system. A model is proposed which can account for the inhibition kinetics. This model is based on two cation-binding sites for which cations compete, a carrier or transporting site, and a second nontransporting (modifier) site with a different array of affinities for cations. The association of certain cations with the modifier site leads to a reduction in the turnover of the carrier, the degree of reduction depending on the cation bound to the modifier site and on the cation being transported.     

Biochimica et biophysica acta,vol. 644 (1981)

In the presence of an iso-osmotic concentration (0.4 M) of LiCl, the exit of cellular K⁺ and concomitant entry of Li⁺ in the marine bacterium, Vibrio alginolyticus, were enhanced by an increase in the medium pH, with an optimum at about pH 9.6. In addition to alkaline pH, the K⁺ exit in the NaCl medium required the presence of a weak base such as diethanolamine, ethanolamine or methylamine, which is permeable to the membrane in its unprotonated form. No net entry of Na⁺ was detected in this case and the amine accumulated in exchange for K⁺. The K⁺ exit observed at alkaline pH could be explained by the function of a K⁺/H⁺ antiporter. Once the cells were loaded with the amine, their exposure to the NaCl medium in the absence of loaded amine induced the entry of Na⁺. In RbCl or CsCl medium, fast entry of Rb⁺ or Cs⁺ and exit of K⁺ were observed at neutral pH (7.5), and the rate of K⁺ exit increased with the medium pH. From these results, we established a simple method for the replcement of cellular cations with a desired cation (Li⁺, Na⁺, K⁺, Rb⁺ or Cs⁺). The present method was found to be applicable also to Escherichia coli.     

Salinomycin: a new monovalent cation ionophore.

The cation discriminations of salinomycin and its derivatives have been studied by measuring complexability with cations and transport rate of them across organic phase. Salinomycin exhibited a great preference for K⁺ over other monovalent and divalent cations in migrating cations into organic phase in two phase systems. The antibiotic mediated the transport of Na⁺ and Rb⁺ as effectively as that of K⁺ across CCl₄ bulk phase, but not those of Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺. From the above results, salinomycin is concluded to act as an alkali ion carrier. The OH-acylated salinomycins retained the activity of parent compound, but the COOH-esterified salinomycins lost the activity.     

Ionic permeation of lipid bilayer membranes mediated by a neutral,noncyclic Li+-selective carrier having imide and ether ligands. I. Selectivity among monovalent cations

Summary We have found that Simon's neutral, noncyclic, Li⁺-selective complexone, which has imide and ether ligands, renders lipid bilayer membranes selectively permeable to certain cations and anions. The present paper characterizes the ability of this molecule to carry monovalent cations; and we show it to be most selective for Li⁺ among the alkali cations, the first reconstitution of Li⁺-selective permeation in lipid bilayer membranes. This complexone acts as an equilibrium-domain carrier for Ag⁺> Li⁺>Tl⁺>Na⁺>NH ₄ ⁺ >Rb⁺>Cs⁺ over a wide range of experimental conditions. The major type of membrane-permeating species formed is a 21 carrier/cation complex dominant except at the lowest salt and carrier concentrations where a 11 carrier/cation, with a similar selectivity sequence, can be detected. Among the groupIa cations the selectivity sequence in bilayers, Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺, is similar to that previously found for this molecule in thick solvent-polymer membrane electrodes. We find this carrier to be more selective to Ag⁺ than to any other monovalent cation yet studied. This high Ag⁺ selectivity is used, together with the dependence of the selectivity on the nature of the N-amide substitutents, to argue that the imide oxygens play a major role as ligands.     

Osmotic swelling of heart mitochondria in acetate and chloride salts. Evidence for two pathways for cation uptake.

Swelling of nonenergized heart mitochondria suspended in acetate salts appears to depend on the activity of an endogenous cation/H⁺ exchanger. Passive swelling in acetate shows a characteristic cation selectivity sequence of Na⁺ >Li⁺ >K⁺, Rb⁺, Cs⁺, or tetramethylammonium, a sharp optimum at pH 7.2–7.3, activation by Ca²⁺, and loss of activity on aging which can be related to loss of endogenous K⁺. The reaction is nearly insensitive to either addition of exogenous Mg²⁺ or removal of membrane Mg²⁺ with EDTA. Each of these characteristics of passive swelling in acetate salts is duplicated in chloride media when tripropyltin is added to induce Cl^?/OH^? exchange. In contrast to nonenergized mitochondria, swelling of respiring mitochondria has been postulated to depend on electrophoretic uptake of cations in response to an interior negative membrane potential. Respiration-dependent swelling in acetate shows an indistinct cation selectivity sequence with Li⁺ and Na⁺ supporting higher rates of swelling at higher efficiency than K⁺, Rb⁺, and Cs⁺. The high rates of respiration-dependent swelling in Li⁺ and Na⁺ are inhibited by low levels of exogenous Mg²⁺ (K_i of 5–10 μm), but a significant swelling with almost no cation selectivity persists in the presences of 2 mm Mg²⁺. Removal of membrane Mg²⁺ by addition of EDTA strongly activates the rate of respiration-dependent swelling and converts a sigmoid dependency of swelling rate on Li⁺ concentration to a hyperbolic one with a Km of about 14 mm Li⁺. The cation selectivity and Mg²⁺ dependence of the reaction induced in chloride salts by tripropyltin are identical to these properties in acetate. Energy-dependent swelling in acetate shows optimum activity at pH 6.5 which appears related to the availability of free acetic acid, since the corresponding reaction induced in chloride shows a broad optimum at about pH 7.5. These studies support the concept that monovalent cations enter nonenergized mitochondria by electroneutral exchange with protons but penetrate respiring mitochondria by electrophoretic movement through one or more uniport pathways. They further suggest that both a Mg²⁺-sensitive uniport with high activity for Na⁺ and Li⁺ and a Mg²⁺-insensitive pathway with little cation discrimination are available in the membrane.     

Activation of wheat germ acetyl CoA carboxylase by potassium and rubidium.

Wheat germ acetyl CoA carboxylase requires certain alkali cations to exhibit maximal activity. Maximal activation results when 60 mM K⁺ or Rb⁺ are included in the assay mixture, whereas only marginal activation occurs in the presence of similar concentrations of Li⁺⁺ and Na⁺⁺. Cs⁺⁺ activates, but less effectively than K⁺ or Rb⁺. Since it is also possible to activate the enzyme maximally using 20 mM potassium isocitrate, but not 20 mM sodium isocitrate, activation of the wheat germ enzyme is due to a cation effect and not to citrate anion.     

Ion complexation in nonactin,monactin, and dinactin: A Raman spectroscopic study

The ability of the macrotetrolide nactins to complex selectivity with a wide variety of cations makes these ionophorous antibiotics important model systems for the study of biologic ionic transport. We report a Raman spectroscopic investigation of the Na⁺, K⁺, Rb⁺, Cs⁺, Tl⁺, NH₄⁺, NH₃OH⁺, C(NH₂)₃⁺, and Ba⁺⁺ complexes of nonactin, monactin, and dinactin in 4:1 (v/v) CH₃OH/CHCl₃ and in the solid state. The nactins display characteristic spectral changes upon complexation, some of which are specific for a given cation. In the K⁺, Rb⁺, Cs⁺, NH₃OH⁺, and C(NH₂)₃⁺ complexes, which are apparently isosteric, the ester carbonyl stretch frequency is found to be linearly proportional to the cation–carbonyl electrostatic interaction energy, as calculated from a simplified model. Deviations for the Na⁺, NH₄⁺, Tl⁺, and Ba⁺⁺ complexes are interpreted as arising from additional nonelectrostatic interactions. Additional information is obtained from other spectral regions and from measurements of depolarization ratios. Spectra of the nactin complexes differ from each other more in the solid state than in solution, reflecting the effects of crystalline contact forces.     

Interactions of alginates with univalent cations

Interactions of alginate with univalent cations in solution have been investigated by circular dichroism (c.d.) and rheological measurements. Poly-l-guluronate chain-segments show substantial enhancement (～ 50%) of c.d. ellipticity in the presence of excess of K⁺, with smaller changes for other univalent cations: Li⁺ < Na⁺ < K⁺ > Rb⁺ > Cs⁺ > NH₄⁺. The maximum c.d. change is attained by 0.3m, with no further increase at higher concentrations of cation. No significant dependence on polymer concentration is observed. Spectral changes for poly-d-mannuronate and heterotypic chain-sequences are much smaller. For intact alginates, the magnitude of c.d. change varies almost linearly with poly-l-guluronate content. Difference spectra (c.d. with excess of univalent counterion minus c.d. in distilled water) can be fitted accurately to two Gaussian bands at 211 and 198 nm, assigned to carboxyl n → π^* and π → π^* transitions, respectively. The perturbations induced by Li⁺, K⁺, Rb⁺, Cs⁺, and NH₄⁺ show a clear family relationship, and are mainly in the π → π^* spectral region. With Na⁺, by contrast, c.d. change is largely confined to the n → π^* transition, and is similar to that previously reported for intermolecular (“egg-box”) binding of divalent cations, consistent with results of rheological studies which indicate Na⁺-induced association of poly-l-guluronate chain-sequences. These associations are further enhanced on freezing and thawing. This combined evidence is interpreted in terms of three modes of interaction between univalent cations and alginate chains in solution: (a) ion-pair formation with carboxyl groups of mannuronate and isolated guluronate residues; (b) specific site-binding to contiguous guluronate residues; and (c) co-operative “egg-box” binding, particularly of Na⁺, between poly-l-guluronate chain-sequences.     

Enniatin B and Valinomycin as Ion Carriers: An Empirical Force Field Analysis

Abstract

The alkali-ion binding properties of two natural depsipeptide ion carriers, enniatin B (EnB) and valinomycin (VM), are examined and compared by the empirical force field method. While VM has been shown to bind preferentially K⁺, Rb⁺, and Cs⁺ over Na⁺ in most solvents, EnB is considerably less specific.

We find that EnB forms two kinds of complexes, internal and external. In internal complexes, the ion binds to all six carbonyl oxygens, while in external ones, only three oxygens, preferentially those of the D-hydroxy-isovaleryl residues, are bound. The size of the internal cavity is best suited for Na⁺, while K⁺ and Rb⁺ squeeze in asymmetrically by distorting the molecule, and Cs⁺ not at all. External binding is much less specific. Since internal complexes possess much higher strain energies than external ones, the latter may be at least as stable as the former, even in fairly non-polar solvents.

VM is calculated to bind only internally, and with much less strain energy than EnB. The size of its internal cavity is well suited for binding the ions K⁺, Rb⁺, and Cs⁺, but is too big for Na⁺. The difference between the binding energies of Na⁺ and K⁺ is much smaller than that between the corresponding hydration enthalpies, thus explaining the binding preference for the latter ion.     

The penetration of some cations into muscle

The influx of Na⁺, K⁺, Rb⁺, and Cs⁺ into frog sartorius muscle has been followed. The results show that a maximum rate is found for K⁺, while Na⁺ and Cs⁺ penetrate much more slowly. Similar measurements with Ca⁺⁺, Ba⁺⁺, and Ra⁺⁺ show that Ba⁺⁺ penetrates at a rate somewhat greater than that of either Ca⁺⁺ or Ra⁺⁺. All these divalent cations, however, penetrate at rates much slower than do the alkali cations. The results obtained are discussed with reference to a model that has been developed to explain the different penetration rates for the alkali cations.     

Alkali metal ion selectivity of the hemocyanin channel

Summary The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K⁺ measured from biionic potentials were: NH ₄ ⁺ (1.52)>Rb⁺ (1.05)>K⁺ (1.0)>Cs⁺ (0.89)>Na⁺ (0.81)>Li⁺ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K⁺, Rb⁺, NH ₄ ⁺ , Cs⁺, Na⁺ and Li⁺, respectively. Current-voltage curves for the different monovalent cations were measured and described using a threebarrier model previously used to explain the voltage dependence of the instantaneous channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH ₄ ⁺ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.     

Ion Specificity of Na+-Transporting Systems in the Plasma Membrane of the Halotolerant Alga Tetraselmis (Platymonas) viridis

Vesicular preparations of plasma membranes (PM) from the microalga Tetraselmis (Platymonas) viridisRouch were used to investigate the ion specificity of the Na⁺/H⁺antiporter and Na⁺-translocating ATPase, two Na⁺-transporting systems previously identified functionally by our studies of T. viridisPM. The Na⁺/H⁺antiporter and Na⁺-ATPase were shown to translocate, with similar efficiencies, Na⁺and Li⁺across the membrane, whereas other cations, such as K⁺, Rb⁺, and Cs⁺, were not transported by these systems. Transport of the latter cations across PM of T. viridisoccurred through the ion channels of PM, which were apparently selective for K⁺.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning α-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of “the cation” in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca²⁺, Mg²⁺ and Mn²⁺) and monovalent metal cations (Li⁺, Na⁺, K⁺ and Cs⁺), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.     

Ion permeation properties of a cloned human 5-HT3 receptor transiently expressed in HEK 293 cells

Summary Human 5-HT₃ receptors expressed in HEK 293 cells were studied using patch-clamp techniques. The permeability ratios of cations to Na⁺ were Li⁺, 1.16; K⁺, 1.04; Rb⁺, 1.11; Cs⁺ 1.11; NMDG⁺, 0.04; Ca²⁺, 0.49, and Mg²⁺, 0.37. The permeability sequence of the alkali metal cations was Lⁱ⁺ > Rb⁺ = Cs⁺ > K⁺ > Na⁺. Increased external concentrations of Ca²⁺ or Mg²⁺ decreased 5-HT-induced currents at all potentials tested in a voltage-independent manner. The single-channel conductance of human 5-HT₃ receptors measured by fluctuation analysis of whole-cell currents was 790 ± 100fS. Differences in the basic properties of 5-HT₃ receptors between species may explain interspecies differences in pharmacological properties.     

Effects of alkali ions on the circadian leaf movements of Oxalis regnellii

The influence of alkali ions on the circadian leaf movements of Oxalis regnellii Mig. was investigated. Ions were given to the oscillating system via the transpiration stream of cut stalks in nutrient medium. Chloride solutions of Rb⁺, Cs⁺, Na⁺ and K⁺ were tested and the results compared to previously published LiCl-results. The period of the circadian leaf movements was unaffected by a continual addition of Na⁺ or K⁺ to the nutrient medium (at least up to 40 mM). Rb⁺, in the concentration of 2.5 or 5 mM, caused a shortening of the period when applied continuously. Rb⁺ concentrations up to 60 mM were tested. Cs⁺ ions caused only lengthenings of the circadian period. Cs⁺ concentrations up to 40 mM were tested. Cs⁺ resembled Li⁺ in producing period lengthenings, but was not as effective as Li⁺ when compared on a concentration basis. Toxicity of the effective ions was in the following order: Li⁺Cs⁺Rb⁺, Rb⁺ pulses (50 mM, 4 h) phase-shifted the rhythm and caused advances. A phase response curve was determined and the maximum steady state advances were of the order of 1 h. The dual effect of the Rb⁺ ions is discussed and is assumed to be due to two counteracting processes, exemplified by Rb⁺-sensitive ATPase-controlled pumping processes and protein synthesis. For comparison, the effects of Rb⁺ and Li⁺ in human depressive disorders is also discussed in relation to their influence on circadian systems. It is emphasized that Rb⁺ and K⁺ behave differently and are not interchangeable in their action on circadian systems.     

Effects of monovalent cations on phosphate accumulation and storage of the ectomycorrhizal fungus Suillus bovinus

Comparative in vivo ³¹P-NMR studies of the fungus Suillus bovinus (L.: Fr.) O. Kuntze in pure culture have produced interesting new data. To investigate the response of phosphate metabolism to a change in external monovalent cations, samples were exposed to a Hoagland solution containing different monovalent cations Li⁺, Na⁺, K⁺, or Rb⁺ at 10 mM concentration. A method of nutrient cycling during analysis where the cation was changed and the phosphate kept constant allowed us to determine the kinetics of phosphate accumulation, storage and incorporation into polyphosphate following exposure to the range of test cations. Different external monovalent cations had different effects upon changes in the content of both phosphate and polyphosphate. Treatment with Li⁺, Na⁺, or Rb⁺ resulted in a change in phosphate accumulation to 60, 73, and 107% and in content of the intracellular mobile polyphosphate (polyP) to 119, 112, and 94%, respectively, compared with the control taken as 100%. The effect of each cation is related to its position in the periodic table. Reversing this process, i.e., exchanging with K⁺, returned phosphate metabolism to normal. Although, the increase in depolarization of the cell membrane should affect the internal pH, fungal metabolism using energy requiring mechanisms appeared necessary to maintain the intracellular pH. Thus, increasing contents of mobile polyP were the consequence of an increasing energy demand. On the other hand, the increasing depolarization of the cell membrane following the sequence Rb⁺ < K⁺ < Na⁺ < Li⁺ inhibited the net P_i accumulation. Furthermore, it is postulated that the P_i accumulation was also regulated by the intracellular content in polyP.     

Effects of monovalent cations on the catalytic activity of pig liver phosphofructokinase.

The monovalent cations NH₄⁺, K⁺, and Rb⁺ activate pig liver phosphofructokinase by increasing the maximal velocity. In the presence of these cations the enzyme retains sigmoid kinetics with respect to fructose-6-phosphate. However, these cations bring about a decrease in the [S]_0.5 for fructose-6-phosphate to an extent directly proportional to their ionic volumes. The apparent dissociation constants of NH₄⁺, K⁺, and Rb⁺ for the enzyme at 0.5 mM ATP and 4 mM Fru6P are 0.2 mM, 8 mM, and 15 mM, respectively. The maximal velocity of the enzyme in the presence of saturating concentrations of Rb⁺ is about 70% of that seen with NH₄⁺ or K⁺. The monovalent cations Li⁺, Na⁺, and Cs⁺ inhibit the enzyme at high concentrations (> 50 mM) by decreasing the maximal velocity. Although the efficiency of inhibition by these cations qualitatively increases with decreasing size, there is no obvious quantitative relationship between efficiency of inhibition and any parameter of ionic size.     

Activation of Rb+ and Na+ uptake into yeast by monovalent cations

⁸⁶Rb⁺ uptake by yeast was not only stimulated by Rb⁺ or K⁺ but also by Na⁺. The uptake of ²²Na⁺ was enhanced by both Rb⁺ and K⁺, but not by Na⁺, which was inhibitory at all concentrations applied. Inhibition of ²²Na⁺ uptake by inactive Na⁺ occurred in two phases: one phase refers to inhibition at low Na⁺ concentrations and the other to inhibition at high Na⁺ concentrations. Our results can be qualitatively described by a two-site transport mechanism, having two cation binding sites, which must be occupied with monovalent cations before transport can occur.