Kinetics of ion transport in lipid membranes induced by lysine-valinomycin and derivatives

Summary Lysine-valinomycin and two N-acyl derivatives are compared with respect to their potency to transport Rb⁺ ions across thin lipid membranes. Lysine-valinomycin acts as a neutral ion carrier only above a pH of about 7 of the aqueous solutions, while at lower pH the molecules seem to be positively charged due to a protonation of the -NH₂ group of the lysine residue.A kinetic analysis based on voltage jump relaxation experiments and on the nonlinearity of the current-voltage characteristics showed that the conductance increment per carrier molecule for uncharged lysine-valinomycin is similar to that of natural valinomycin. The attachment of a rather bulky side group such as the dansyl or para-nitrobenzyloxycarbonyl group reduced by approximately one order of magnitude.Some of the relaxation data of the valinomycin analogues were influenced by an unspedfic relaxation of the pure lipid membrane. This structural relaxation represents a limitation to the possibility of analyzing specific transport systems in thin lipid membranes by the voltage jump or charge pulse techniques. It is shown that the time dependence of this structural relaxation — which was first published by Sargent (1975) — is at variance with a three capacitor equivalent circuit of the membrane, which was suggested by Coster and Smith (1974) on the basis of a.c. measurements. A modified equivalent circuit has been found to represent a satisfactory analogue for the current relaxation in the presence of valinomycin. It turned out, however, that such an equivalent circuit provides little insight into the molecular mechanism of transport.     

Temperature-jump experiments on thin lipid membranes in the presence of valinomycin

Summary Temperature jump relaxation experiments on planar lipid membranes in the presence of valinomycin were performed using the absorption of a strong light flash as an energy source for the generation of the T-jump. The relaxation of the current carried by valinomycin/Rb⁺ complexes was measured. The results were interpreted on the basis of a transport model which was also analyzed by voltage jump relaxation experiments. The study shows that the application of the T-jump technique provides valuable information about transport kinetics as well as the dynamics of the membrane structure. At the given experimental conditions the relaxation of the current is believed to reflect a temperature-dependent transition of the membrane to a new conformational state of lower order. The relaxation could be resolved with the present technique only at low temperatures and for membranes of high microviscosity.     

Mechanism of ion transport through lipid bilayer-membranes mediated by peptide cyclo-(d-Val-l-Pro-l-Val-d-Pro)3

The cyclic dodecapeptide PV, cyclo-(d-Val-l-Pro-l-Val-d-Pro)₃, a structural analogue of the ion-carier valinomycin, increase the cation permeability of lipid bilayer membranes. This paper reports the results of two types of relaxation experiments, namely relaxation of the membrane current after a voltage jump and decay of the membrane voltage after a charge pulse in lipid bilayer membranes exposed to PV. From the relaxation data, the rate constant for the translocation of the ion carrier complex across the membrane, as well as the partition coefficient of the complex between water and membrane solution interface were computed and found to be about one order of magnitude less than the comparable values for valinomycin (Val). Furthermore, the dependence of the initial membrane conductivity on ion concentration was used to evaluate the equilibrium constant, K, of complexation between PV and some monovalent cations in water. The values of K yield the following selectivity sequence of PV: Na⁺ < NH₄⁺ < K⁺ < Cs⁺ < Rb⁺. These and earlier results are consistent with the idea that PV promotes cation movement across membranes by the solution complexation mechanism which involves complexation between ion and carrier in the aqueous phase and transport of the carrier across the membrane. In the particular form of the solution complexation mechanism operating here, the PV present in the PV-cation complex carrying charge across the membrane derives from the side from which the current is flowing (cis-mechanism). As shown previously, valinomycin, in contrast to PV, acts by an interfacial complexation mechanism in which the Val in the Val-cation complex derives from the side toward which current is flowing (trans-mechanims). The comparison of the kinetic properties of these two closely related compounds yields interesting insights into the relationship between chemical structure and function of ion carriers.     

Valinomycin-mediated ion transport through neutral lipid membranes: Influence of hydrocarbon chain length and temperature

Summary Stationary electrical conductance experiments together with nonstationary relaxation experiments allow a quantitative determination of rate constants describing carrier-mediated ion transport. Valinomycin-induced ion transport across neutral lipid membranes was studied. The dependence of the transport parameters on the chain length of the lipid molecules, on the kind of alkali ion, and on the temperature was determined. The relaxation time the current following a voltage jump shows a marked increase with decreasing temperature or with increasing chain length of the lipid molecules. This variation of is interpreted on the basis of a varying membrane fluidity. It is shown that under favorable circumstances the equilibrium constant of complex formation in the aqueous phase may be obtained from membrane experiments. Furthermore, the kinetics of exchange of valinomycin between membrane and water was studied. We found a marked influence of the totus surrounding the black film on the kinetics as well as on the total amount of valinomycin molecules in the membrane. The problem of location of the free carrier molecules inside the membrane is discussed.     

Ion transport mediated by the valinomycin analogue cyclo(L-Lac-L-Val-D- Pro-D-Val)3 in lipid bilayer membranes

Cyclo(L-Lac-L-Val-D-Pro-D-Val)3 (PV-Lac) a structural analogue of the ion-carrier valinomycin, increases the cation permeability of lipid bilayer membranes by forming a 1:1 ion-carrier complex. The selectively sequence for PV-Lac is identical to that of valinomycin; i.e., Rb+ greater than K+ greater than Cs+ greater than or equal to NH+4 greater than Na+ greater than Li+. The steady-state zero-voltage conductance, G(0), is a saturating function of KCl concentration. A similar behavior was found for Rb+, Cs+, and NH+4. However, the ion concentration at which G(0) reaches a plateau strongly depends on membrane composition. The current-voltage curves present saturating characteristics, except at low ion concentrations of Rb+, K+, or Cs+. The ion concentration at which the saturating characteristics appear depends on membrane composition. These and other results presented in this paper agree with a model that assumes complexation between carrier and ion at the membrane-water interface. Current relaxation after voltage-jump studies were also performed for PV-Lac. Both the time constant and the amplitude of the current after a voltage jump strongly depend on ion concentration and membrane composition. These results, together with the stationary conductance data, were used to evaluate the rate constants of the PV-Lac-mediated K+ transport. In glycerolmonooleate they are: association rate constant, 2 x 10(6) M-1 s-1; dissociation rate constant, 4 x 10(5) s-1; translocation rate constant for complex, 5 x 10(4) s-1; and the rate of translocation of the free carrier (ks), 55 s-1. ks is much smaller for PV-Lac than for valinomycin and thus limits the efficiency with which the carrier is able to translocate cations across the membrane.     

Kinetics of macrotetrolide-induced ion transport across lipid bilayer membranes

Ion transport across lipid bilayer membranes in the presence of macrotetrolide antibiotics has been studied by stationary conductance and nonstationary relaxation methods. The results are discussed on the basis of a carrier model which has already been successfully applied to valinomycin induced ion transport. Again a kinetic analysis has been performed from which the single rate constants of the carrier model could be derived. In addition the equilibrium constant of complex formation in the aqueous phase could be determined. Measurements have been made for 4 macrotetrolides, for several ions and for various chain lengths of the lipids molecules composing the membrane.     

Leucine transport in plasma membrane vesicles of Saccharomyces cerevisiae

Yeast plasma membrane vesicles were obtained by the fusion of liposomes with purified yeast membranes by means of the freeze thaw-sonication technique. Beef heart mitochondria cytochrome-c oxidase was incorporated into the vesicles. Addition of substrate (ascorbate/TMPD/cytochrome c) generated a membrane potential negative inside, and an alkaline pH gradient inside the vesicle, that served as the driving force for leucine transport. Both delta pH and delta psi could drive leucine transport. When delta pH was increased in the presence of valinomycin and potassium, at the expense of delta psi, leucine uptake increased by 10%.     

Determination of rate constants in carrier-mediated diffusion through lipid bilayers

In this work data are presented on the relaxation current, under a voltage step, through soybean lipid bilayers in the presence of the carrier valinomycin. Measurements of voltage-dependent steady-state conductance have also been performed. These measurements are sufficient to calculate the full set of kinetic parameters determining the transport.The data are analyzed according to the kinetic model, based on an Eyring treatment of the carrier-mediated diffusion. Complementary measurements of conductance as a function of antibiotic concentration have also been reported. These data allow one to calculate the membrane-solution partition coefficient of the carrier and the surface charge density of the membrane. The results are compared with those previously obtained with membranes of different lipid composition.     

Temperature-jump method for studying the fast transport of Na+ by (221) C10-cryptand across lipid membranes.

The kinetics of Na+ transport by (221)C10-cryptand through thin lipid membranes were determined by performing temperature-jump relaxation experiments on large unilamellar vesicles (L.U.V.) loaded with a fluorescent pH indicator. Applying temperature jumps of 4 to 7 degrees C to liposomes having phosphate as internal buffer and Tris as external buffer resulted in transmembrane delta pH's of about 0.104 to 0.182. After a temperature-jump, a decay in the delta pH was observed which corresponded to a Na+/H+ exchange occurring through membranes in the simultaneous presence of the cryptand and a proton carrier. The transport of Na+ ions by (221)C10 was found to be a fast kinetic process. Its initial rate increased with both the temperature and the cryptand concentrations. In addition, the temperature-induced changes in the apparent rate constants of the translocation of Na+ by (221)C10 were carrier concentration-dependent, and the apparent activation energy required to activate the transport decreased significantly with increasing cryptand concentrations. The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes.     

Control of proteoliposomal cytochrome c oxidase: the overall reaction

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (delta psi) and pH gradient (delta pH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse delta psi and increase delta pH; the respiration rate decreases. High levels of valinomycin, however, decrease delta pH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases delta psi and collapses delta pH; the respiration rate increases. On a millivolt equivalent basis delta pH is a more effective inhibitor of activity than is delta psi. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both delta pH and delta psi components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing delta pH but in a nonlinear way to delta psi ("non-ohmically"). High levels of both delta psi and delta pH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both delta pH and delta psi components of the proton-motive force, but is more sensitive to delta pH than to delta psi at an equivalent delta mu H+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella.     

Temperature jump as a new technique to study the kinetics of fast transport of protons across membranes

Application of a temperature jump (2.5 degrees C) to a suspension of liposomes, having phosphate (delta pK/delta T approximately 0.005) as the internal buffer and tris(hydroxymethyl)aminomethane (delta pK/delta T approximately 0.031) as the external buffer, created a delta pH (pHin - pHout) of positive sign in ca. 5 microseconds. Decay of this delta pH was monitored by using the fluorescent pH indicator 8-hydroxy-1,3,6-pyrenetrisulfonic acid entrapped inside the liposome. This technique is useful to study transmembrane proton movement in the time range 5 microseconds-10 s at physiological pH values. The kinetics of proton transport aided by ion carriers such as nigericin, monensin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and valinomycin were studied by our method. The electrogenic nature of transport by CCCP and valinomycin and electroneutral ion transport by nigericin and monensin were shown. From the kinetics of proton transport aided by gramicidin, the time-averaged single-channel conductance of gramicidin channels was estimated to be (2.1 +/- 0.5) X 10(-16) S for H+ at pH 7.5.     

Temperature-dependent effects of cholesterol on sodium transport through lipid membranes by an ionizable mobile carrier

Temperature-jump relaxation experiments on Na⁺ transport by (221)C₁₀-cryptand were carried out in order to study the influence of cholesterol and its temperature-dependence on ion transport through thin lipid membranes. The experiments were performed on large, negatively charged unilamellar vesicles (LUV) prepared from mixtures of dioleoylphosphatidylcholine, phosphatidic acid and cholesterol (mole fractions 0–0.43), at various temperatures and carrier concentrations. The initial rates of Na⁺ transport and the apparent rate constants of its translocation by (221)C₁₀ increased with the carrier concentration and the temperature. The incorporation of cholesterol into the membranes significantly reduced the carrier concentration- and temperature-dependence of these two parameters. The apparent energy required to activate the transport decreased significantly with increasing carrier concentrations at any given cholesterol molar fraction, and increased significantly with the cholesterol molar fraction at any given carrier concentration. Our interpretation of the action of cholesterol on this transport system is based on the assumption that the binding cavity of cryptands is likely to be located towards the aqueous side of the dipole layer. The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes, and of the interactions occurring between an ionizable mobile carrier and the membrane.     

ATP-dependent uptake of 5-hydroxytryptamine by secretory granules isolated from thyroid parafollicular cells.

The current study was done to test the hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than on the membrane potential component of the proton electrochemical gradient across the granular membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents, such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from secretagogue-stimulated parafollicular cells is greater than that into granules isolated from unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due to an increase in delta pH.     

Optical and electrical studies on dansyllysine-valinomycin in thin lipid membranes

Summary Dansyllysine-valinomycin, a fluorescent analogue of the ionophore valinomycin was synthesized and incorporated into black lipid membranes. Its concentration inside the membrane was measured fluorometrically and was also determined from electrical relaxation experiments, which were analyzed on the basis of a previously proposed carrier model. The results of both methods agreed within less than one order of magnitude. This appears satisfactory in view of the sources of error inherent in both procedures.A conductance increment per carrier molecule of about 3 · 10^{–17 –1} was obtained for dansyllysine-valinomycin in diphytanoyllecithin membranes at 25 C and 1M RbCl in the aqueous phases. This is about 400 times smaller compared to unmodified valinomycin in monoolein membranes. The difference is mainly caused by the change in the membrane properties and to a smaller extent by the structural modification of the carrier.     

The Rate Constants of Valinomycin-Mediated Ion Transport through Thin Lipid Membranes

Electrical relaxation experiments have been performed with phosphatidylinositol bilayer membranes in the presence of the ion carrier valinomycin. After a sudden change of the voltage a relaxation of the membrane current with a time constant of about 20 μsec is observed. Together with previous stationary conductance data, the relaxation amplitude and the relaxation time are used to evaluate the rate constants of valinomycin-mediated potassium transport across the lipid membrane. It is found that the rate constants of translocation of the free carrier S and the carrier-ion complex MS⁺ are nearly equal (2·10⁴ sec^-1) and are of the same order as the dissociation rate constant of MS⁺ in the membrane-solution interface (5·10⁴ sec^-1). The equilibrium constant of the heterogeneous association reaction M⁺ (solution) + S (membrane) → MS⁺ (membrane) is found to be ~ 1 M^-1, about 10⁶ times smaller than the association constant in ethanolic solution.     

Temperature-dependent effects of cholesterol on sodium transport through lipid membranes by an ionizable mobile carrier.

Temperature-jump relaxation experiments on Na+ transport by (221)C10-cryptand were carried out in order to study the influence of cholesterol and its temperature-dependence on ion transport through thin lipid membranes. The experiments were performed on large, negatively charged unilamellar vesicles (LUV) prepared from mixtures of dioleoylphosphatidylcholine, phosphatidic acid and cholesterol (mole fractions 0-0.43), at various temperatures and carrier concentrations. The initial rates of Na+ transport and the apparent rate constants of its translocation by (221)C10 increased with the carrier concentration and the temperature. The incorporation of cholesterol into the membranes significantly reduced the carrier concentration- and temperature-dependence of these two parameters. The apparent energy required to activate the transport decreased significantly with increasing carrier concentrations at any given cholesterol molar fraction, and increased significantly with the cholesterol molar fraction at any given carrier concentration. Our interpretation of the action of cholesterol on this transport system is based on the assumption that the binding cavity of cryptands is likely to be located towards the aqueous side of the dipole layer. The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes, and of the interactions occurring between an ionizable mobile carrier and the membrane.     

Evidence for nucleotide-dependent passive H+ transport protein in the plasma membrane of barley roots

Plasma membranes were isolated from barley roots by two-phase partitioning, and octylglucoside-soluble and -insoluble fractions were obtained. The insoluble fractions were reconstituted into liposomes, and the plasma membrane H(+)-ATPase was shown to participate in MgATP-dependent H(+) transport activity. The H(+) transport was decreased when the octylglucoside-soluble fraction was reconstituted together with the insoluble fraction. The decrease was not due to inhibition of the H(+)-ATPase, but rather was likely due to the increased H(+) leakage from the proteoliposome. The octylglucoside-soluble fraction was, therefore, reconstituted in the liposomes and the passive H(+) transport was determined using the pH jump method. A pH gradient across the membranes was generated by the pH jump, and the gradient was found to be dissipated by passive H(+) transport. The H(+) transport required ATP, K(+), and valinomycin. The H(+)-transport also occurred when ADP, AMP, GTP, or ATP-gamma-S was present instead of ATP, and did not occur when the octylglucoside-soluble fraction was boiled before the reconstitution. These findings suggest that nucleotide-dependent H(+ )transport protein is present in the plasma membrane of root cells.     

An extended kinetic analysis of valinomycin-induced Rb-transport through monoglyceride membranes

Summary The time course of the current following a voltage jump, which is applied to monoglyceride bilayers in the presence of valinomycin, shows two relaxation times. This is basically in agreement with a simple carrier model which has been described in full detail formerly. Relaxation times and amplitudes allow a calculation of the rate constants of the transport model. The presented data supplement an analysis which was hitherto based only on the slower relaxation process and on information derived from the nonlinearity of currentvoltage characteristics. The additional resolution of the faster relaxation time allowed an approximate determination of the voltage dependence of the translocation rate constant for the carrier-ion-complex and provided evidence for a small voltage dependence of the interfacial reaction. The dependence of the relaxation parameters on the ion concentration in the aqueous phase was interpreted assuming a saturation of the ion concentration at the reaction plane at high bulk concentrations.     

An extended kinetic analysis of valinomycin-induced Rb-transport through monoglyceride membranes.

The time course of the current following a voltage jump, which is applied to monoglyceride bilayers in the presence of valinomycin, shows two relaxation times. This is basically in agreement with a simple carrier model which has been described in full detail formerly. Relaxation times and amplitudes allow a calculation of the rate constants of the transport model. The presented data supplement an analysis which was hitherto based only on the slower relaxation process and on information derived from the nonlinearity of current-voltage characteristics. The additional resolution of the faster relaxation time allowed an approximate determination of the voltage dependence of the translocation rate constant for carrier-ion-complex and provided evidence for a small voltage dependence of the interfacial reaction. The dependence of the relaxation parameters on the ion concentration in the aqueous phase was interpreted assuming a saturation of the ion concentration at the reaction plane at high bulk concentrations.     

Impedance Analysis of Ion Transport Through Supported Lipid Membranes Doped with Ionophores: A New Kinetic Approach

Kinetics of facilitated ion transport through planar bilayer membranes are normally analyzed by electrical conductance methods. The additional use of electrical relaxation techniques, such as voltage jump, is necessary to evaluate individual rate constants. Although electrochemical impedance spectroscopy is recognized as the most powerful of the available electric relaxation techniques, it has rarely been used in connection with these kinetic studies. According to the new approach presented in this work, three steps were followed. First, a kinetic model was proposed that has the distinct quality of being general, i.e., it properly describes both carrier and channel mechanisms of ion transport. Second, the state equations for steady-state and for impedance experiments were derived, exhibiting the input–output representation pertaining to the model’s structure. With the application of a method based on the similarity transformation approach, it was possible to check that the proposed mechanism is distinguishable, i.e., no other model with a different structure exhibits the same input–output behavior for any input as the original. Additionally, the method allowed us to check whether the proposed model is globally identifiable (i.e., whether there is a single set of fit parameters for the model) when analyzed in terms of its impedance response. Thus, our model does not represent a theoretical interpretation of the experimental impedance but rather constitutes the prerequisite to select this type of experiment in order to obtain optimal kinetic identification of the system. Finally, impedance measurements were performed and the results were fitted to the proposed theoretical model in order to obtain the kinetic parameters of the system. The successful application of this approach is exemplified with results obtained for valinomycin–K⁺ in lipid bilayers supported onto gold substrates, i.e., an arrangement capable of emulating biological membranes.