Lanthanide-ion transport across phospholipid vesicular membranes: a comparison of alamethicin 30 and A23187 using1H-NMR spectroscopy

The kinetics of Pr³⁺ transport by the ionophores alamethicin 30 and A23187 across unilamellar phospho-lipid vesicular membranes has been compared by following the time-dependent changes in the¹H-NMR spectrum of the vesicles. The measured rates of transport allow stoichiometries of the transporting species to be deduced which are consistent with channel- and carrier-mediated mechanisms respectively. The method provides a useful complement to planar bilayer conductivity studies of these systems.     

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187.

The antibiotic A23187 carries Ca2+ across Müller-Rudin membranes made from 1,2-dierucoyl-sn-glycero-3-phosphocholine and n-decane. The conductance of the membranes is not increased by the Ca2+-transport. The flux depends linearly on Ca2+ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4-5 between pH 6 and pH 8. Maximal Ca2+-fluxes of about 10(-10) mol-cm-2-s-1 were found. A counter transport of H+ could not be detected. The complex formation between A23187 and Ca2+ in egg phosphotidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2:1 complex. Optical absorption measurements on single phophatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.     

Fluorescence study of the divalent cation-transport mechanism of ionophore A23187 in phospholipid membranes

The mechanism for transport of divalent cations across phospholipid bilayers by the ionophore A23187 was investigated. The intrinsic fluorescence of the ionophore was used in equilibrium and rapid-mixing experiments as an indicator of ionophore environment and complexation with divalent cations. The neutral (protonated) form of the ionophore binds strongly to the membrane, with a high quantum yield relative to that in the aqueous phase. The negatively charged form of the ionophore binds somewhat less strongly, with a lower quantum yield, and does not move across the membrane. Complexation of the negatively charged form with divalent cations was measured by the decrease in fluorescence. An apparent rate constant (kapp) for transport of the ionophore across the membrane was determined from the rate of fluorescence changes observed in stopped-flow rapid kinetic experiments. The variation of kapp was studied as a function of pH, temperature, ionophore concentration, membrane lipid composition, and divalent cation concentration and type. Analysis and comparison with equilibrium constants for protonation and complexation show that A23187 and its metal:ionophore complexes bind near the membrane-water interface in the lipid polar-head region. The interfacial reactions occur rapidly, compared with the transmembrane reactions, and are thus in equilibrium during transport. The transport cycle can be described as follows: a 1:1 complex is formed between the membrane bound A23187-(Am-) and the aqueous divalent cation with dissociation constant K1 approximately 4.6 x 10(-4) M. This is in equilibrium with a 1:2 (metal:ionophore) complex (K2 approximately 3.0 x 10(-4) [ionophore/lipid]) that is responsible for transporting the divalent cations across the membrane. The rate constant for translocation of the 1:2 complex is 0.1-0.3 s-1. Dissociation of the complex of the trans side and protonation occur rapidly. The rate constant for translocation of H+ . A23187- is 28 s-1. A theory is presented that is capable of reproducing the kinetic data at any calcium concentration. The cation specificity for ionophore complex transport (kapp), determined at low ionophore concentration for a series of divalent cations, was found to be proportional to the equilibrium constant for 1:1 complexation. The order of ion specificity for these processes was found to be Ca2+ greater than Mg2+ greater Sr2+ greater than Ba2+. Interactions with Na+ were not observed. Maximal values of kapp were observed for vesicles prepared from pure dimyristoyl phosphatidylcholine. Inclusion of phosphatidyl ethanolamine, phosphatidic acid, or dipalmatoyl phosphatidylcholine resulted in lower values of kapp. Calcium transport by A23187 is compared with that of X537A, and it is shown that the former is 67-fold faster. The difference in rates is due to differences in the ability of each ionophore to form a 1:2 complex from a 1:1 complex.     

Carboxylic ionophore (lasalocid A and A23187) mediated lanthanide ion transport across phospholipid vesicles

The transport kinetics of three lanthanide ions (viz., Pr3+, Nd3+, and Eu3+) across dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine unilamellar vesicles mediated by the two carboxylic ionophores lasalocid A and A23187 have been studied by proton nuclear magnetic resonance spectroscopy. Time-dependent changes in the chemical shifts of head group choline signals have been measured to calculate apparent rate constants of transport. These experiments have been done at different ionophore concentrations to determine the stoichiometry of the transporting species. The rates of transport have been found to be faster in the absence of intravesicular La3+ compared to those observed in its presence. The stoichiometry of the transporting species has been found to be 2:1 (ionophore:cation) for both lasalocid A and A23187 in dimyristoylphosphatidylcholine vesicles. However, stoichiometries of greater than 2 have been obtained for lasalocid A mediated lanthanide ion transport across dipalmitoylphosphatidylcholine vesicles. Possible reasons for the observations of such noninteger stoichiometries are discussed. Our results also indicated that A23187 is a more efficient carrier ionophore than lasalocid A.     

Indole-3-acetic acid-mediated transport of Mn2+ and other ions across phosphatidylinositol vesicular membranes as determined by 31P-NMR.

The indolic plant hormone, indole-3-acetic acid (IAA), mediated the transport of Mn2+ and other ions into small unilamellar vesicles prepared from soybean phosphatidylinositol (PI) and this process has been studied using 31P nuclear magnetic resonance spectroscopy (NMR). The rate of Mn2+ movement into PI vesicles is dependent on IAA concentration and temperature with an IAA stoichiometry of 4.1 and an activation energy of 16.8 kcal mol-1 derived for the rate-determining process. These values are altered by low concentrations of endogenous ions (which can be removed by treatment with EDTA) present in the PI. With non-EDTA-treated PI, values of 2.3 and 23.0 kcal mol-1 were obtained for the stoichiometry and activation energy, respectively. These values indicate that (a) IAA interacts with PI membranes; (b) IAA-induced changes in membrane permeability can be substantially modulated by ions and (c) IAA very significantly influences the rate of movement of some (but possibly not all) cations across PI membranes. Such effects are also modified by the oxidation state of the PI.     

Transport of mono- and divalent cations across chloroplast membranes mediated by the ionophore A23187

Effects of the ionophore A23187 on isolated broken and intact chloroplasts in the pH range of 6.2 to 7.6 have been studied. In both types of chloroplasts, uncoupling of photosynthetic electron transport by A23187 (6–10 μm) was mediated either by Mg²⁺ or—in the absence of divalent cations (i.e., when EDTA was added to the medium)—by high concentrations of Na⁺, but not of K⁺ ions. At increased concentrations of the ionophore (above about 10 μm) and high pH (7.2 to 7.6), uncoupling in broken chloroplasts was also mediated by K⁺ ions. The inhibition of the energy-dependent slow decline of chlorophyll fluorescence in intact chloroplasts by the ionophore (which denotes uncoupling) is reversed by EDTA in the presence of K⁺, but not of Na⁺ ions. In 3-(3′,4′-dichlorophenyl)1,1-dimethylurea-poisoned intact chloroplasts, the yield of variable chlorophyll fluorescence is lowered by A23187 + EDTA and increased again by addition of NaCl or KCl. Chlorophyll fluorescence spectra at 77 °K of intact chloroplasts incubated with A23187 + EDTA indicated that the distribution of excitation energy had changed in favor of photosystem I, as expected from a depletion of Mg²⁺. This change was reversed by MgCl²⁺, KCl, or NaCl. From a comparison of low-temperature fluorescence spectra of broken and intact chloroplasts at different levels of Mg²⁺ in the medium, the concentration of free Mg²⁺ in the stroma of the intact chloroplasts at pH 7.6 in the dark was estimated at 1 to 4 mm. The results show that in chloroplasts the specificity of A23187 for divalent cations is limited. In the presence of EDTA, the ionophore mediates fast $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange across thylakoid membranes, whereas K⁺ is transferred much less efficiently. Both Na⁺ and K⁺ ions seem to be transported readily across the chloroplast envelope by the action of the ionophore, leading to an exchange of Mg²⁺ for monovalent cations at the thylakoid membrane surfaces in intact chloroplasts.     

NMR kinetic studies of the ionophore X-537A-mediated transport of manganous ions across phospholipid bilayers.

Nuclear magnetic resonance spectroscopy has been applied as a method for studying manganous ions transport across the membrane of phosphatidylcholine vesicles. The rates of the ionophore X-537A (lasalocid A)-mediated Mn2+ transport have been measured as a function of ionophore concentration, pH of the vesicle suspension, and temperature. The translocation was found to occur via a neutral complex composed of one manganous ion bound in two ionized X-537A molecules (Mn X2). The activation energy for the overall transport process was determined to be 22 +/- 5 kcal/mol. Also a pKa of 5.0 +/- 0.2 was determined for the ionophore acid dissociation equilibrium in the vesicle suspension.     

NMR kinetic studies of the ionophore X-537A-mediated transport of manganous ions across phospholipid bilayers

Nuclear magnetic resonance spectroscopy has been applied as a method for studying manganous ions transport across the membrane of phosphatidylcholine vesicles. The rates of the ionophore X-537A (lasalocid A)-mediated Mn²⁺ transport have been measured as a function of ionophore concentration, pH of the vesicle suspension, and temperature. The translocation was found to occur via a neutral complex composed of one manganous ion bound to two ionized X-537A molecules (Mn X₂). The activation energy for the overall transport process was determined to be 22 ± 5 kcal/mol. Also a pK_a of 5.0 ± 0.2 was determined for the ionophore acid dissociation equilibrium in the vesicle suspension.     

Enhancement of the muscarinic synaptosomal phospholipid labeling effect by the ionophore A23187

Addition of either acetylcholine (ACh) or the ionophore A23187 to synaptopsomes resulted in a selective stimulation of 32Pi incorporation into phosphatidate (PhA) and phosphatidylinositol (PhI), while the labeling of phosphatidylinositol phosphate (PhIP) and phosphatidylinositol diphosphate (PHIP2) was reduced. The inclusion of both ACh and A23187 resulted in a synergistic increase in PhA and PhI labeling, and a synergistic decrease in the labeling of the polyphosphoinositides. Added calcium was not required, although inclusion of EGTA prevented the alterations in lipid labeling. The enhanced labeling of PhA and PhI by ACh or A23187 was not the result of either an increase in the radioactivity of the precursor [32P]ATP pool, or increased de novo synthesis of these lipids as judged from the incorporation of [3H]glycerol, [3H]glucose or [3H]myo-inositol. The synergistic alterations in PhA, PhI, and polyphosphoinositide labeling were observed with ionophore only in the presence of selected muscarinic agonists, and with the inclusion of atropine or scopolamine the labeling reverted to a value which approximated that seen with the ionophore alone. Synergistic effects on phospholipid labeling with muscarinic agonists were also obtained with the calcium ionophore, ionomycin, but not with X537A, monensin, or valinomycin. Neither the apparent number of muscarinic receptors present, nor their affinity for the ligand were altered by the presence of A23187. In prelabeling experiments, A23187 accelerated the loss of [32P]label from PhIP and PhIP2, and the rate of loss was further augmented by the addition of ACh. Neither agent produced comparable effects on the breakdown of prelabeled PhA or PhI. It is suggested that phosphodiesteratic cleavage of the polyphosphoinositides might account for both the decrease in labeled PhIP and PhIP2 and increased labeling of PhA and PhI via the availability of resultant diglyceride. In any event, the results demonstrate that the turnover of polyphosphoinositides, in addition to that of PhA and PhI, is linked to the activation of muscarinic receptors.     

Two mechanisms of H+/OH- transport across phospholipid vesicular membrane facilitated by gramicidin A.

Two rate-limiting mechanisms have been proposed to explain the gramicidin channel facilitated decay of the pH difference across vesicular membrane (delta pH) in the pH region 6-8 and salt (MCI, M+ = K+, Na+) concentration range 50-300 mM. 1) At low pH conditions (approximately 6), H+ transport through the gramicidin channel predominantly limits the delta pH decay rate. 2) At higher pH conditions (approximately 7.5), transport of a deprotonated species (but not through the channel) predominantly limits the rate. The second mechanism has been suggested to be the hydroxyl ion propogation through water chains across the bilayer by hydrogen bond exchange. In both mechanisms alkali metal ion transport providing the compensating flux takes place through the gramicidin channels. Such an identification has been made from a detailed study of the delta pH decay rate as a function of 1) gramicidin concentration, 2) alkali metal ion concentration, 3) pH, 4) temperature, and 5) changes in the membrane order (by adding small amounts of chloroform to vesicle solutions). The apparent activation energy associated with the second mechanism (approximately 3.2 kcal/mol) is smaller than that associated with the first mechanism (approximately 12 kcal/mol). In these experiments, delta pH was created by temperature jump, and vesicles were prepared using soybean phospholipid or a mixture of 94% egg phosphatidylcholine and 6% phosphatidic acid.     

Alterations in the vesicular pattern and wall growth ofPhycomyces induced by the calcium ionophore A 23187

Summary Hyphal elongation, chitin synthesis in vivo, and invertase secretion inPhycomyces blakesleeanus were all inhibited almost instantly by the addition of 5–10 M calcium ionophore A 23187. Protein biosynthesis was inhibited in these conditions by 30–50%. The ionophore did not affect cell respiration for at least 40 min. Effect on chitin biosynthesis was not due to alterations of the chitin synthetase levels or its activity; nor to impairement in GlcNAc metabolism. In drug-treated cells the number of apical vesicles was severely reduced even at very short periods of incubation, and these low numbers remained constant for at least 60 min of incubation with the ionophore. We suggest that the ionophore collapses the cellular calcium gradient and/or interferes with the normal electrical transhyphal current. As a consequence, formation and migration of apical vesicles are inhibited. These results are further evidence of the role of vesicles in fungal tip growth and exhibit the fact that active chitin synthetase is short-lived in vivo demanding its continuous supply by chitosomes to the cell surface.Abbreviations GlcNAc N-acetylglucosamine - TCA trichloroacetic acid - UDPGIcNAc uridine diphosphate-N-acetylglucosamine - DMSO dimethylsulfoxide     

Monensin-mediated transports of H+, Na+, K+ and Li+ ions across vesicular membranes: T-jump studies.

Theoretical expression for the rate of decay of delta pH across vesicular membrane due to carrier-mediated ion transports, 1/tau, has been modified taking note of carrier states (such as mon- and mon-H-M+) for which the translocation rate constants in the membrane are small. The rates of delta pH decay due to monensin-mediated H+ and M+ transports (M+ = Na+, K+, Li+) observed in our experiments in the pH range 6-8, and [M+] range 50-250 mM at 25 degrees C have been analysed with the help of this expression. delta pH across soybean phospholipid vesicular membranes were created by temperature jump in our experiments. The following could be inferred from our studies. (a) At low pH (approximately 6) 1/tau in a medium of Na+ is greater than that in a medium of K+. In contrast with this, at higher pH (approximately 7.5) 1/tau is greater in a medium of K+. Such contradictory observations could be understood with the help of our equation and the parameters determined in this work. The relative concentrations of the rate-limiting species (mon-H, mon-K, and mon-Li at Ph approximately 7 in vesicle solutions having Na+, K+ and Li+, respectively) can explain such behaviours. (b) The proton dissociation constant KH for mon-H in the lipid medium (pKH approximately 6.55) is larger than the reported KH in methanol. (c) The concentrations of mon- and mon-H-Na+ are not negligible under the conditions of our experiments. The latter species cause a [Na+]-dependent inhibition of ion transports. (d) The relative magnitudes of metal ion dissociation constants KHM (approximately 0.05 M) for mon-H-Na+ and KM (approximately 0.03 M) for mon-Na suggest that the carboxyl group involved in the protonation may not be dominantly involved in the metal ion complexation. (e) The estimates of KM (approximately 0.03 M for Na+, 0.5 M for K+ and 2.2 M for Li+) follow the ionophore selectivity order. (f) The rate constants k1 and k2 for the translocations of mon-H and mon-M (M+ = Na+, K+ and Li+) are similar in magnitude (approximately 9 x 10(3) s-1) and are higher than that for nig-H and nig-M (approximately 6 x 10(3) s-1) which can be expected from the relative molecular sizes of the ion carriers.     

Effect of ethylmaleimide on the transport of Ca+ and K+ ions across mitochondrial membranes

As already reported, it has been found that the gradient of protons, set up across the inner membrane during the Ca2+ uptake by rat liver mitochondria, can be completely reversed by the addition of NEM. Identical results have been obtained by following the energy dependent K+ uptake. In these last conditions, the rate of H+ efflux supported by succinate oxidation is greatly enhanced only when NEM is added after rotenone. It is proposed that the increased rate other than to the inhibition of Pi uptake, as suggested by Reynafarje and Lehninger, could also be ascribed to a further decrease in the energetic level of the membrane as well as to an increased rate of succinate-Pi exchange diffusion reaction induced by NEM. A possible direct effect of NEM on succinate oxidation has been also considered to account for the inhibition observed when it is added before rotenone.     

Change in the physical state of platelet plasma membranes upon ionophore A23187 activation. A fluorescence polarization study

Human platelets were isolated and fluorescence-labelled by 1,6-diphenylhexatriene. Diphenylhexatriene was essentially localized in the plasma membrane, as indicated by trinitrobenzenesulfonate-quenching experiments. A decrease of the fluorescence polarization of diphenylhexatriene was observed upon ionophore A23187 addition in the absence of aggregation. 0.3 microM ionophore allowed to reach the maximum rate of the decrease of fluorescence polarization; it also maximally stimulated the light transmission change, the serotonin release and the thromboxane B2 synthesis. The amplitude of the fluorescence polarization decrease was maximum at platelet concentrations between 4 X 10(7) and 7 X 10(7)/ml. The presence of Ca2+ in the medium increased the rate constant of the polarization change. Chlorpromazine (60 microM) completely inhibited this transition, but at 30 microM its inhibitory effect was reversed by Ca2+. The membrane events implied in platelet activation very likely lead to fluidization of the plasma membrane, perhaps by its fusion with the membranes of internal granules which are relatively depleted of cholesterol. Ca2+ plays a central role in the triggering of the observed effects at the membrane level.     

Role of phospholipid hydrolysis in the mechanism of toxic cell death by calcium and ionophore A23187

Manganese chloride inhibits the hydrolysis of arachidonate-containing phospholipids stimulated in 3T3 mouse fibroblasts by ionophore A23187 in the presence of extracellular calcium. The inhibition is reduced by increasing extracellular calcium concentrations. Stimulation by A23187 of this phospholipid hydrolysis and cell killing are inhibited at similiar concentrations by (i) manganese chloride or (ii) reduced extracellular calcium. These results indicate an important role for the phospholipid hydrolysis in the mechanism of cell killing by A23187 plus calcium. Analysis of the rates of the two processes indicates that phospholipid hydrolysis triggers cell killing, but it is not itself the membrane permeabilizing step.     

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane

Multilamellar phospholipid vesicles are introduced into the cis compartment on one side of a planar phospholipid bilayer membrane. The vesicles contain a water-soluble fluorescent dye trapped in the aqueous phases between the lamellae. If a vesicle containing n lamellae fuses with a planar membrane, an n-1 lamellar vesicle should be discharged into the opposite trans compartment, where it would appear as a discernible fluorescent particle. Thus, fusion events can be assayed by counting the number of fluorescent particles appearing in the trans compartment. In the absence of divalent cation, fusion does not occur, even after vesicles have been in the cis compartment for 40 min. When CaCl2 is introduced into the cis compartment to a concentration of greater than or equal to 20 mM, fusion occurs within the next 20 min; it generally ceases thereafter because of vesicle aggregation in the cis compartment. With approximately 3 x 10(8) vesicles/cm3 in the cis compartment, about 25-50 fusion events occur following CaCl2 addition. The discharge of vesicular contents across the planar membrane is the most convincing evidence of vesicle-membrane fusion and serves as a model for that ubiquitous biological phenomenon--exocytosis.     

Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187

Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current.On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca²⁺ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca²⁺, Mg²⁺, or Mn²⁺ are detected. A23187 produces fluorescent transients under conditions of Ca²⁺ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca²⁺ content. Changes in A23187 fluorescence due to mitochondrial Ca²⁺ accumulation are much smaller by comparison and fluorescence transients are not detected.Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca²⁺ and Mg²⁺, and less than 2 nsec in the presence of excess EDTA.The present results are consistent with a model of ionophore-mediated cation transport in which free M²⁺ binds with A23187 at the membrane surface to form the complex M(A23187)⁺. Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187)₂, which carries the divalent cation through the membrane.These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.