Kinetics of ion translocation across charged membranes mediated by a two-site transport mechanism. Effects of polyvalent cations upon rubidium uptake into yeast cells.

(1) The effect of surface charge upon the kinetics of monovalent cation translocation via a two-site mechanism is investigated theroretically. (2) According to the model dealt with, typical relations are expected for the dependence of the kinetic parameters of the translocation process upon the concentration of a polyvalent cation, differing essentially from those derived for the case in which the membrane carries no excess charge. (3) Even when a polyvalent cation does not compete with the substrate cation for binding to the translocation sites, apparently competitive inhibition may occur when the membrane is negatively charged. (4) The model is tested experimentally by studying the effects of the polyvalent cations Mg2+, Sr2+, Ca2+, Ba2+ and Al3+ upon Rb+ uptake into yeast cells at pH 4.5 A good applicability is found. (5) Equimolar concentrations of polyvalent cations reduce the rate of the Rb+ uptake into yeast cells in the order Mg2+ less than Sr2+ less than Ca2+ less than Ba2+ less than Al3+. (6) The conclusion is reached that the reduction in the rate of Rb+ uptake caused by the polyvalent cations applied results mainly from screening of the negative fixed charges on the membrane surface and binding to these negative sites rather than competition with Rb+ for the transport sites. (7) The results of our investigation indicate the affinity of the alkaline-earth cations for the negative fixed charges on the surface to the yeast cell membrane increases in the orther Mg2+ less than Sr2 less than Ca2+ less than Ba2+. (8) Probably mainly phosphoryl groups determine the net charge on the membrane of the yeast cell at a medium pH of 4.5.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH, cations and phosphate.

The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Cyclic nucleotide-gated channels of rat olfactory receptor cells: divalent cations control the sensitivity to cAMP

cAMP-gated channels were studied in inside-out membrane patches excised from the apical cellular pole of isolated olfactory receptor cells of the rat. In the absence of divalent cations the dose-response curve of activation of patch current by cAMP had a KM of 4.0 microM at -50 mV and of 2.5 microM at +50 mV. However, addition of 0.2 or 0.5 mM Ca2+ shifted the KM of cAMP reversibly to the higher cAMP concentrations of 33 or 90 microM, respectively, at -50 mV. Among divalent cations, the relative potency for inducing cAMP affinity shifts was: Ca2+ > Sr2+ > Mn2+ > Ba2+ > Mg2+, of which Mg2+ (up to 3 mM) did not shift the KM at all. This potency sequence corresponds closely to that required for the activation of calmodulin. However, the Ca(2+)-sensitivity is lower than expected for a calmodulin-mediated action. Brief (60 s) transient exposure to 3 mM Mg2+, in the absence of other divalent cations, had a protective effect in that following washout of Mg2+, subsequent exposure to 0.2 mM Ca2+ no longer caused affinity shifts. This protection effect did not occur in intact cells and was probably a consequence of patch excision, possibly representing ablation of a regulatory protein from the channel cyclic nucleotide binding site. Thus, the binding of divalent cations, probably via a regulatory protein, controls the sensitivity of the cAMP-gated channels to cAMP. The influx of Ca2+ through these channels during the odorant response may rise to a sufficiently high concentration at the intracellular membrane surface to contribute to the desensitization of the odorant- induced response. The results also indicate that divalent cation effects on cyclic nucleotide-gated channels may depend on the sequence of pre-exposure to other divalent cations.     

Chemical basis for selectivity of metal ions by the Bacillus subtilis cell wall.

The use of equilibrium dialysis techniques established that isolated cell walls of Bacillus subtilis possess selective affinities for several cations. The binding of these cations to the cell wall was influenced by the presence of various functional groups in the peptidoglycan matrix. Selective chemical modification of the free carboxyl and amino groups showed that when amino groups were replaced by neutral, bulky, or negatively charged groups, the sites available for cation complexing generally increased. Introduction of positive charges into the wall resulted in a marked decrease in the numbers of metal binding sites and usually a decrease in the apparent association constants. Both teichoic acid and peptidoglycan contribute to the sites available for interaction with metals. Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity. Competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls. When the hydrogen ion concentration was increased, the affinity of the walls for metals decreased, but the numbers of metal binding sites remained constant, suggesting that cations and protons also compete for the same sites.     

Differential interaction of cations with the thiamine and biotin transport proteins of Lactobacillus casei

Lactobacillus casei cells contain separate and specific binding proteins which mediate the cellular uptake of thiamine and biotin. In buffered salt solutions, these proteins exhibit a very high affinity for their vitamin substrate. Dissociation constants (Kd values) at pH 7.5 are 0.03 and 0.15 nM for thiamine and biotin, respectively. Optimal binding of biotin requires the presence of cations. This cation dependence is substantial since the Kd for biotin is 60-fold higher in a buffer containing 0.1 mM K-Hepes, compared with a buffer composed of 50 mM K-Hepes and 5 mM MgCl2. Measurements of Kd versus cation concentration showed that Mg2+ is 300-fold more effective than K+ in promoting biotin binding. The extent of cation dependence decreases as the pH is reduced from 7.5 to 5.0, suggesting that protons can partially fulfill the cation requirement. In contrast, binding of thiamine to the thiamine transport protein shows no dependence on the ionic composition of the medium. These results suggest that the transport protein for the anionic vitamin, biotin, contains a binding site for cations. Cotransport of both the vitamin and cation into the cell might then occur during the normal transport cycle, allowing the cellular uptake of the vitamin to occur against the membrane potential. Conversely, the cationic vitamin, thiamine, does not appear to be transported into the cell as a complex with other ions.     

Characterization of metal ion-binding sites in bacteriorhodopsin

We have investigated the effects of the binding of various metal ions to cation-free bacteriorhodopsin ("blue membrane"). The following have been measured: shift of the absorption maximum from 603 to 558 nm (blue to purple transition), binding isotherms, the release of H+ upon binding, and the decay of the deprotonated intermediate of the photocycle, M412. We find that all cations of the lanthanide series, as well as the alkali and alkali earth metals earlier investigated, are able to bring about the absorption shift, whereas Hg2+ and Pt4+ are not. Sigmoidal spectroscopic titration curves and nonsigmoidal binding curves suggest that there are two high affinity sites for cations in bacteriorhodopsin. Binding to the site with the second highest affinity is responsible for the absorption shift. Divalent cation binding to blue membrane causes release of about six protons, whereas higher numbers of protons are released by trivalent cations, suggesting that the shift of absorption maximum involves proton release from carboxyl group(s). The metal ion bound to this site must be surrounded by carboxyl oxygen atoms acting together as a multidentate ligand with a specific geometry because multivalent ions are effective only when capable of octahedral coordination. Lanthanide ions dramatically inhibit M412 decay at pH above 6.3, an effect probably due to binding to lipid phosphoryl groups.     

Binding of polycationic antibiotics and polyamines to lipopolysaccharides of Pseudomonas aeruginosa.

Polycations, such as aminoglycoside and peptide antibiotics, and naturally occurring polyamines were found to bind to the lipopolysaccharide of Pseudomonas aeruginosa and alter its packing arrangement. Binding of cations was measured by the displacement of a cationic spin probe from lipopolysaccharide into the aqueous environment upon addition of competitive cations. The level of probe displacement was dependent on the concentration and charge of the competing cation, with the more highly charged cations being more effective at displacing probe. The relative affinity of several antibiotics for lipopolysaccharide correlated with their ability to increase outer membrane permeability, while the relative affinity of several polyamines correlated with their ability to stabilize the outer membrane. Probe mobility within the lipopolysaccharide head group was shown to be decreased by cationic antibiotics and unaltered or increased by polyamines. We propose that antibiotic permeability and disruption of outer membrane integrity by polycationic antibiotics results from binding of the antibiotic to anionic groups on lipopolysaccharide with a consequent change in the conformation of lipopolysaccharide aggregate structure.     

Calcium-sensitive univalent cation channel formed by lysotriphosphoinositide in bilayer lipid membranes.

A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb+ greater than Cs+ greater than Na+ greater than K+ greater than Li+. The conductance of the membrane was increased up to a value of about 10-2 ohm-1/cm2 with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn+ greater than Cd2+ greater than Ca2+ greater than Sr2+ greater than Mg2+.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH,cations and phosphate

The uptake of Ca²⁺ and Sr²⁺ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics.The rate of Ca²⁺ and Sr²⁺ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca²⁺ and Sr²⁺ at low pH may be explained by a decrease of the surface potential.The inhibition of Ca²⁺ and Sr²⁺ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane.Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Tests of an electrostatic screening hypothesis of the inhibition of neurotransmitter release by cations at the frog neuromuscular junction.

We have investigated an electrostatic screening hypothesis of cationic inhibition of quantal release at the neuromuscular junction of the frog (Rana pipiens). According to this hypothesis, increasing the extracellular concentration of an inhibitory cation reduces the quantal content (m) of the end-plate potential by reducing the ability of negative surface charge to attract Ca2+ to the external surface of the presynaptic membrane. The inhibitory power of various cations should depend only on their net ionic charge and should increase strongly with increasing charge. We have demonstrated, in Ringer's solutions containing modified concentrations of Na+, Ca+, and Mg2+, that at fixed concentrations of Ca2+ and Na+ (a) the dependence of m on [Mg2+]0 is satisfactorily accounted for by electrostatic theory and (b) the dependence of m on the univalent cation concentration of the modified Ringer's solution is satisfactorily predicted from the Mg2+ inhibition of m. (Glucosamine or arginine was used to replace a fraction of the Na+ content of Ringer's solution in the latter experiments.) These results are consistent with electrostatic screening actions of Mg2+ and univalent cations in the inhibition of m. We have also re-examined the inhibition of m caused by the addition to Ringer's solution of two trace concentration divalent cations, Mn2+ and Sr2+. Our data suggest that the inhibition of m by Sr2+ at high quantal contents may also be due to surface charge screening, while the potent inhibitory actions of Mn2+ may be due to its ability to bind negative surface charge.     

Mechanism and Role of Divalent Cation Binding of Bacteriorhodopsin

Several observations have already suggested that the carboxyl groups are involved in the association of divalent cations with bacteriorhodopsin (Chang et al., 1985). Here we show that at least part of the protons released from deionized purple membrane (`blue membrane') samples when salt is added are from carboxyl groups. We find that the apparent pK of magnesium binding to purple membrane in the presence of 0.5 mM buffer is 5.85. We suggest this is the pK of the carboxyl groups shifted from their usual pK because of the proton concentrating effect of the large negative surface potential of the purple membrane. Divalent cations may interact with negatively charged sites on the surface of purple membrane through the surface potential and/or through binding either by individual ligands or by conformation-dependent chelation. We find that divalent cations can be released from purple membrane by raising the temperature. Moreover, purple membrane binds only about half as many divalent cations after bleaching. Neither of these operations is expected to decrease the surface potential and thus these experiments suggest that some specific conformation in purple membrane is essential for the binding of a substantial fraction of the divalent cations. Divalent cations in purple membrane can be replaced by monovalent, (Na⁺ and K⁺), or trivalent, (La⁺⁺⁺) cations. Flash photolysis measurements show that the amplitude of the photointermediate, O, is affected by the replacement of the divalent cations by other ions, especially by La⁺⁺⁺. The kinetics of the M photointermediate and light-induced H⁺ uptake are not affected by Na⁺ and K⁺, but they are drastically lengthened by La⁺⁺⁺ substitution, especially at alkaline pHs. We suggest that the surface charge density and thus the surface potential is controlled by divalent cation binding. Removal of the cations (to make deionized blue membrane) or replacement of them (e.g. La⁺⁺⁺-purple membrane) changes the surface potential and hence the proton concentration near the membrane surface. An increase in local proton concentration could cause the protonation of critical carboxyl groups, for example the counter-ion to the protonated Schiff's base, causing the red shift associated with the formation of both deionized and acid blue membrane. Similar explanations based on regulation of the surface proton concentration can explain many other effects associated with the association of different cations with bacteriorhodopsin.     

Interactions of permeant cations with sodium channels of squid axon membranes.

To determine how the permeant cations interact with the sodium channel, the instantaneous current-voltage (I-V) relationship, conductance-ion concentration relationship, and cation selectivity of sodium channels were studied with internally perfused, voltage clamped squid giant axons in the presence of different permeant cations in the external solution. In Na-containing media, the instantaneous I-V curve was almost linear between +60 and -20 mV, but deviated from the linearity in the direction to decrease the current at more negative potentials. The linearity of instantaneous I-V curve extended to more negative potentials with lowering the external Ca concentration. The I-V curve in Li solution was almost the same as that in Na solution. The linearity of the I-V curve improved in NH4 solution exhibiting only saturation at -100 mV with no sign of further decrease in current at more negative potentials. Guanidine and formamidine further linearized the instantaneous I-V curve. The conductance of the sodium channels as measured from the tail current saturated at high concentrations of permeant cations. The apparent dissociation constants determined from the conductance-ion concentration curve at -60 mV were as follows: Na, 378 mM; Li, 247 mM; NH4, 174 mM; guanidine, 111 mM; formamidine, 103 mM. The ratio of the test cation permeability to the sodium permeability as measured from the reversal potentials of tail currents varied with the test cation concentration and/or the membrane potential. These observations are incompatible with the independence principle, and can be explained on the basis of the Eyring's rate theory. It is suggested that the slope of the instantaneous I-V curve is determined by the relative affinity of permeant cations and blocking ions (Ca) for the binding site in the sodium channel. The ionic selectivity of the channel depends on the energy barrier profile of the channel.     

The kinetics and divalent cation inhibition of plasma membrane ATPase in the yeast Candida albicans

The kinetics of ATP hydrolysis and cation effects on ATPase activity in plasma membrane from Candida albicans ATCC 10261 yeast cells were investigated. The ATPase showed classical Michaelis-Menten kinetics for the hydrolysis of Mg X ATP, with Km = 4.8 mM Mg X ATP. Na+ and K+ stimulated the ATPase slightly (9% at 20 mM). Divalent cations in combination with ATP gave lower ATPase activity than Mg X ATP (Mg greater than Mn greater than Co greater than Zn greater than Ni greater than Ca). Divalent cations inhibited the Mg X ATPase (Zn greater than Ni greater than Co greater than Ca greater than Mn). Free Mg2+ inhibited Mg X ATPase weakly (20% inhibition at 10 mM). Computed analyses of substrate concentrations showed that free Zn2+ inhibited Zn X ATPase, mixed (Zn2+ + Mg2+) X ATPase, and Mg X ATPase activities. Zn X ATP showed high affinity for ATPase (Km = 1.0 mM Zn X ATP) but lower turnover (52%) relative to Mg X ATP. Inhibition of Mg X ATPase by (free) Zn2+ was noncompetitive, Ki = 90 microM Zn2+. The existence of a divalent cation inhibitory site on the plasma membrane Mg X ATPase is proposed.     

Mass-Action Expressions of Ion Exchange Applied to Ca, H, K, and Mg Sorption on Isolated Cells Walls of Leaves from Brassica oleracea

The cation exchange properties of cell walls isolated from collard (Bassica oleracea var acephala D.C.) leaves were investigated. Cation sorption on cell walls was described by mass-action expressions of ion exchange, rather than by the traditional Donnan equilibrium. The mass-action expressions enable the selectivity of the wall for one cation over another to be determined unambiguously from ion exchange isotherms. We found that: (a) the cation composition of the wall varied as a function of the solution cation concentration, solution cation composition, and pH in a way predicted by mass action; (b) the affinity of the wall for divalent cations increased as the equivalent fraction of divalent cation on the wall increased, and as the concentration of divalent cations in solution increased; (c) the selectivity of the wall for any metal cation pair was not altered by the concentration of H⁺ in solution or on the wall; (d) H⁺ sorption on the wall may be treated as a cation exchange reaction making it possible to calculate the relative affinity of the wall for metal cation pairs from H⁺-metal (Me) titration curves; and (e) the relative affinity of the wall for the cations we studied was: H⁺ (K⁺ ≥ Ca²⁺) > Mg²⁺. A cation-exchange model including surface complexes is consistent with observed cation selectivity. We conclude that metal cations interact with the wall to minimize or eliminate long-range electrostatic interactions and suggest that this may be due to the formation of site-specific cation-wall surface complexes.     

Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ antiporters with different functions in cell physiology

The family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.     

External monovalent cations that impede the closing of K channels

We have examined the effects of a variety of monovalent cations on K channel gating in squid giant axons. The addition of the permeant cations K, Rb, or Cs to the external medium decreases the channel closing rate and causes a negative shift of the conductance-voltage relationship. Both of these effects are larger in Rb than in K. The opening kinetics of the K channel are, on the other hand, unaffected by these monovalent cations. Other permeant species, like NH4 and Tl, slightly increase the closing rate, whereas the relatively impermeant cations Na, Li, and Tris have little or no effect on K channel gating. The permeant cations have different effects on the reversal potential and the shape of the instantaneous current-voltage relationship. These effects give information about entry and binding of the cations in K channels. Rb, for example, enters the pore readily (large shift of the reversal potential), but binds tightly to the channel interior, inhibiting current flow. We find a correlation between the occupancy of the channel by a monovalent cation and the closing rate, and conclude that the presence of a monovalent cation in the pore inhibits channel closing, and thereby causes a leftward shift in the activation-voltage curve. In causing these effects, the cations appear to bind near the inner surface of the membrane.     

Mechanism of electrogenic cation transport by the cloned organic cation transporter 2 from rat

The organic cation transporter 2 (OCT2) is expressed in plasma membranes of kidney and brain. Its transport mechanism and substrates are debated. We studied substrate-induced changes of electrical current with the patch clamp technique after expression of rat OCT2 in oocytes. Activation of current, corresponding to efflux, was observed for small organic cations, e.g. choline. In contrast, the bigger cations quinine and tetrabutylammonium elicited no change in current. However, transport of choline could be inhibited by applying quinine or tetrabutylammonium to the cytoplasmic side. Inhibition of organic cation efflux by quinine was competitive with substrates. Quinine at the inside also inhibited substrate influx from the outside. Current-voltage analysis showed that both maximal turnover and apparent affinity to substrates are voltage-dependent. Substrate-induced currents with organic cations on both membrane sides reversed as predicted from the Nernst potential. Our results clearly identify the electrochemical potential as driving force for transport at neutral pH and exclude an electroneutral H(+)/organic cation(+) exchange. We suggest the existence of an electroneutral organic cation(+) exchange and propose a model for a carrier-type transport mechanism.     

Phosphoglycolate phosphatase. Effect of cation and pH on activity.

P-glycolate phosphatase requires divalent cations for activity. Activity-pH curves identified 2 active site residues with pK values at pH 5.7 and pH 9.1 in the presence of magnesium and at pH 5.7 and pH 7.5 in the presence of manganese or cobalt. Saturation velocity kinetics enabled the identification of two distinct divalent cation binding sites. The first, nonspecific site has a K0.5 of 2 to 7 x 10(-5) M, depending on the cation and the pH. The second site, which is specific for magnesium, binds this cation in a negatively cooperative fashion. The affinity at pH 8.1 varies approximately 100-fold from the first magnesium bound to the fourth. The negative cooperativity is greatest at high pH. Because the pH range of activity is very broad, both the phosphate monoanion and dianion of P-glycolate must be bound as the substrate. The concentration of these two species at the apparent Km is independent of magnesium concentration. The P-glycolate.magnesium complex is kinetically inactive.     

Role of carboxyl residues and membrane lipids in cation binding to bacteriorhodopsin

To investigate the site specificity of cation binding to bacteriorhodopsin, carboxyl groups were chemically modified in purple membrane preparations from Halobacterium halobium. Cation binding followed by EPR and visible spectroscopy has led us to the conclusion that two cations bind to the surface regions and that at least one cation binds to carboxyl groups in the protein interior. Conformational freedom is necessary for the cooperative conversion of deionized blue species to cation-reconstituted purple species. Studies of white membranes from the JW-5 strain showed that a higher content of charged lipids results in the binding of approximately 100 more color-regulating cations and in negative cooperativity in the blue-to-purple species conversion. A greater dependence of protein structure on these bound cations suggests a role for cations in the modulation of opsin-lipid interaction.