Kinetic analysis of carrier-mediated ion transport by the charge-pulse technique

Summary The charge-pulse technique has been used previously for the study of quasistationary processes in membranes which required only a moderate time resolution. It is shown here that a time resolution of about 400 nsec may be achieved with this technique and that it may be applied to the kinetic analysis of carrier-mediated ion transport. By this method we have studied the transport of alkali ions through optically black monoolein membranes in the presence of the ion carrier valinomycin. All three relaxation processes that are predicted by theory have been resolved. From the relaxation times and the relaxation amplitudes the rate constants for the association (k _R ) and the dissociation (k _D ) of the ioncarrier complex, as well as the translocation rate constants of the complex (k _MS ) and the free carrier (k _S ) could be obtained. For 1m Rb⁺ at 25° C the values arek _R =3×10⁵ m ^–1 sec^–1,k _D =2×10⁵ sec^–1,k _MS =3×10⁵ sec^–1,k _S =4×10⁴ sec^–1. The activation energies of the single rate constants which have been estimated from experiments at two different temperatures range between 50 and 90 kJ/mol.     

Non-equilibrium voltage noise generated by ion transport through pores

In this paper, we describe a systematic approach to the theoretical analysis of non-equilibrium voltage noise that arises from ions moving through pores in membranes. We assume that an ion must cross one or two barriers in the pore in order to move from one side of the membrane to the other. In our analysis, we consider the following factors: a) surface charge as a variable in the kinetic equations, b) linearization of the kinetic equations, c) master equation approach to fluctuations. To analyze the voltage noise arising from ion movement through a two barrier (i.e., one binding site) pore, we included the effects of ions in the channel's interior on the voltage noise. The current clamp is considered as a white noise generating additional noise in the system. In contrast to what is found for current noise, at low frequencies the voltage noise intensity is reduced by increasing voltage across the membrane. With this approach, we demonstrate explicity for the examples treated that, apart from additional noise generated by the current clamp, the non-equilibrium voltage fluctuations can be related to the current fluctuations by the complex admittance.     

Current noise generated by electrogenic ion pumps

Active ion transport by ATP-or light-driven pumps involves a sequence of elementary steps such as binding and release of ions, as well as conformational transitions of the pump protein. At the microscopic level the individual reaction steps occur at random intervals, and therefore the current generated by electrogenic pumps fluctuates around a mean value. In this paper, a theoretical treatment of the electrical noise associated with active ion transport is given. The analysis, which is based on the calculation of the correlation function, yields the spectral intensity S ₁ of current noise as a function of frequency, f. The shape of S _I(f) contains information on the rate constants as well as on the magnitude of the charge displacements occuring during single reaction steps. The contribution of electrogenic pumps to the total voltage noise of the cell may be estimated from S _I(f) and from the frequency-dependent impedance of the cell membrane.     

Effect of 3-phenylindole on lipophilic ion and carrier-mediated ion transport across bilayer lipid membranes

The physical effects of 3-phenylindole, an antimicrobial compound which interacts with phospholipids, on ion transport across phosphatidylcholine-cholesterol bilayers have been investigated using three lipophilic ions and one ion-carrier complex. It was found that 3-phenylindole increased membrane electrical conductance of positively charged membrane probes and decreased electrical conductance of negatively charged probes. The enhancement of conductance detected by nonactin-K+ complex and tetraphenylarsonium+ was several orders of magnitude, whereas the suppression of conductance due to tetraphenylborate- and dipicrylamine- was less than a factor of ten. Presence of 3-phenylindole in aqueous phase slightly decreased adsorption of tetraphenylborate- and dipicrylamine- at the membrane surface. From the voltage dependence of the steady-state conductance it was shown that 3-phenylindole induced kinetic limitation of membrane transport of potassium mediated by nonactin. No such limitation was found in the case of tetraphenylarsonium+ transport. These results are shown to be consistent with the present concept of ion diffusion in membranes and the assumption that 3-phenylindole decreases the electric potential in the membrane interior. The asymmetry of the effect of 3-phenylindole on the magnitude of conductance changes for positively and negatively charged membrane permeable ions is also discussed as a reflection of the discreteness of both the absorbed 3-phenylindole and lipid dipoles.     

Presteady-state kinetics and carrier-mediated transport: A theoretical analysis

Summary Kinetic studies of cotransport mechanisms have so far been limited to the conventional steady-state approach which does not allow in general to resolve either isomerization or ratelimiting steps and to determine the values of the individual rate constants for the elementary reactions involved along a given transport pathway. Such questions can only be answered using presteady-state or relaxation experiments which, for technical reasons, have not yet been introduced into the field of cotransport kinetics. However, since two recent reports seem compatible with the observation of such transient kinetics, it would appear that theoretical studies are needed to evaluate the validity of such claims and to critically evaluate the expectations from a presteady-state approach. We thus report such a study which was performed on a simple four-state mechanism of carrier-mediated transport. The time-dependent equation for zero-trans substrate uptake was thus derived and then extended to models withp intermediary steps. It is concluded that (p-1) exponential terms will describe the approach to the steady state but that such equations have low analytical value since the parameters of the flux equation cannot be expressed in terms of the individual rate constants of the elementary reactions for models withp>5. We thus propose realistic simplifications based on the time-scale separation hypothesis which allows replacement of the rate constants of the rapid steps by their equilibrium constants, thereby reducing the complexity of the kinetic system. Assuming that only one relaxation can be observed, this treatment generates approximate models for which analytical expressions can easily be derived and simulated through computer modeling. When performed on the four-state mechanism of carrier-mediated transport, the simulations demonstrate the validity of the approximate solutions derived according to this hypothesis. Moreover, our approach clearly shows that presteady-state kinetics, should they become applicable to (co)transport kinetics, could be invaluable in determining more precise transport mechanisms.     

Autocorrelation analysis of hydrophobic ion current noise in lipid bilayer membranes.

The autocorrelation function of a given process is related to its spectral density by the Wiener-Khintchine theorem, and both expressions contain the same information. We report here a measurement of the current noise produced in a lipid bilayer membrane doped with hydrophobic anions of dipicrylamine. The results are in good agreement both with relaxation measurements on the same membrane and with an analysis of the spectral density of the current noise for this system which has been presented by other workers. Although measurement of the spectral density function is generally more complete for technical reasons, the autocorrelation function provides, for the case studied here, more physical insight into the underlying charge transport mechanism. We find that the measured autocorrelation function is negative at short, but nonzero, times. This is a consequence of the operative conductance mechanism in this case, which cannot carry current continuously in the same direction without compensatory reverse flow.     

Influence of membrane structure on the kinetics of carrier-mediated ion transport through lipid bilayers

Charge-pulse relaxation experiments of valinomycin-mediated Rb⁺ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experimental data the rate constants of association (k_R) and dissociation (k_D) of the ion-carrier complex as well as the rate constants of translocation of the complex (k_MS) and of the free carrier (k_S) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants k_S and k_MS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C₂₀ fatty acid from one to four resulted in an increase of k_S by a factor of seven and in an increase of k_MS by a factor of twenty-four. The stability constant K = k_R/k_D of the ion-carrier complex as well as the translocation rate constants k_S and k_MS were found to depend strongly on the nature of the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced k_R, k_MS and k_S up to seven-fold.     

Kinetics of carrier-mediated ion transport in two new types of solvent-free lipid bilayers.

In contrast with the usual glyceryl-monooleate/decane (GMO-D) bilayer lipid membranes, new membranes, formed from a mixture of GMO in squalene (GMO-S) or from a mixture of GMO in triolein (GMO-T), seem to be almost solvent free. Our results from voltage-jump relaxation studies, using these "solvent-free" membranes with the homologue carriers, nonactin, monactin, dinactin, trinactin, and tetranactin, are compared with the corresponding ones for GMO-D membranes. With all homologues, solvent-free membranes show an increase of the free carrier translocation rate, ks, by a factor of 2.5, a decrease in the dissociation rate constant of the complex, kDi, by a factor of 1.5 and no significant change in its formation rate constant, kRi. However, the principal effect of the absence of solvent in these membranes is an increase by a factor of approximately 10 of the translocation rate constant for moving the complex across the membrane, kis. This increase varies regularly from a factor of 7-15 with decreasing carrier size, and is always larger for GMO-T than for GMO-S membranes. These solvent-free effects are interpreted in terms of modifications of electrostatic and hydrophobic energy profiles in the membrane.     

Light-induced, carrier-mediated transport of tetracycline by Rhodopseudomonas sphaeroides.

Tetracycline accumulation by the phototrophic bacterium Rhodopseudomonas sphaeroides has been studied, using the fluorescence properties of the antibiotic and measuring uptake of [7- 3H]tetracycline. Accumulation was carrier mediated, with a Km of approximately 300 micronM. Efflux also appeared to be carried mediated, with a Km of 25 mM. Chlorotetracycline competitively inhibited tetracycline transport. The transport was energy dependent. Efflux occurred during the influx process, and an energy-requiring steady state was reached when influx balanced efflux. Transport was inhibited by metabolic inhibitors such as antimycin A, cyanide, and iodoacetate. Proton conductors such as carbonylcyanide m-chlorophenyl hydrazone were strongly inhibitory. Efflux was not energy dependent. Efflux is partially blocked by mercuric ions and completely blocked by an external pH of 9 to 11. Although efflux rates increased continuously with lowering of the pH, influx rates have a sharp maximum at pH 7.     

Nonequilibrium voltage fluctuations in biological membranes. II. Voltage and current noise generated by ion carriers, channels and electrogenic pumps

As applications of the general theoretical framework of charge transport in biological membranes and related voltage and current noise, a number of model calculations are presented for ion carriers, rigid channels, channels with conformational substates and electrogenic pumps. The results are discussed with special reference to the problem of threshold values for sensory transduction processes and their limitations by voltage fluctuations. Furthermore, starting from the special results of model calculations, an attempt is made to determine more general aspects of electric fluctuations generated by charge-transport processes in biological membranes: different frequency dependences of voltage and current noise, and dependence of noise intensities with increasing distance from the equilibrium state.     

The specificity of carrier-mediated auxin transport by suspension-cultured crown gall cells

1. The specificity of the auxin transport system of suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch- is examined with regard to 2,4-Dichlorophenoxyacetic acid (2,4 D), l-Naphthylacetic acid (NAA) and Benzoic acid (BA) as well as for indole-3-acetic acid (IAA). — 2. All four weak acids can be accumulated by the cells from a medium more acidic than the cytoplasm. This is by virtue of non-specific passive diffusion of their lipid-soluble protonated forms down a concentration gradient. The corresponding anionic species are much less permeant. The extent of the accumulation is dependent on the pH difference that is maintained by the cells between their cytoplasm and the incubation medium. Studies of the concentration dependence of BA and NAA net uptake at a series of external pHs suggest that an acidification of the cytoplasm can be eventually brought about by the entry of weak acid into the cells. — 3. The uptake of 2,4 D, as well as that of IAA, has a saturable carrier-mediated component in addition to the passive diffusion of the undissociated acid. These saturable components probably represent anion uptake and appear to be mediated by a common carrier. The kinetic studies provided no evidence for the participation of carriers in the transport of BA or NAA. — 4. It was shown that the efflux of 2,4 D also has a carrier-mediated component and it is suggested that both the influx and efflux of IAA and 2,4 D occur on a common carrier. — 5. The inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA), stimulates the net uptake of IAA by inhibiting carrier-mediated efflux of IAA from the cells. However, TIBA could not be demonstrated to have a significant effect on 2,4 D transport and any perturbation that occurs is very small in comparison with its effect on IAA movement. To account for this, the proposed common carrier could exhibit some difference in its internal binding characteristics betweend 2,4 D and IAA. An alternative explanation is that a second carrier is present, which mediates IAA efflux only, and which is inhibited by TIBA. — 6. TIBA has no significant effect on the transport of either BA or NAA, except to bring about an inhibition of net uptake, and a corresponding stimulation of efflux, when it is present at concentrations sufficient to acidify the cytoplasm. —7. The crown gall cells are compared to intact plant tissues capable of polar auxin transport with regard to the specificities exhibited for the transport of the auxins IAA, 2,4 D and NAA and the non-auxin BA.Abbreviations IAA indol-3-yl acetic acid - 2,4 D 2,4-Dichlorophenoxyacetic acid - NAA 1-Naphthylacetic acid - BA Benzoic acid - TIBA 2,3,5-triiodobenzoic acid     

Evidence for carrier-mediated transport of glucocorticoids by human placental membrane vesicles

Glucocorticoid uptake by isolated placental membrane vesicles has been studied in an attempt to identify a membrane-mediated carrier mechanism. A preliminary communication from this laboratory has reported that uptake of the glucocorticoid corticosterone by these vesicles was a time-dependent, saturable, osmotically sensitive process (Fant, M.E., Harbison, R.D. and Harrison, R.W. (1979) J. Biol. Chem. 254, 6218-6221), but did not conclusively demonstrate a carrier mechanism. Further studies of labeled corticosterone uptake by placental vesicles are described herein which indicate that steroid uptake by these vesicles is a carrier-mediated process. We found that corticosterone uptake was temperature-sensitive, and an apparent phase-transition effect on the rate of uptake was seen to occur at approximately 16 degrees C. Treatment of the vesicles with phospholipase A2 and the sulfhydryl group attacker, p-chloromercuriphenylsulfonate, inhibited corticosterone uptake. In contrast to our previous findings in intact cells, neuraminidase treatment of membranes did not inhibit steroid uptake, perhaps indicating a species variation. Lastly, it was possible to show that corticosterone movement across the membrane exhibited countertransport, a phenomenon common only to carrier-mediated transport mechanisms. These studies show that placental vesicles accumulate corticosterone by a carrier-mediated mechanism.     

Hydrogen ion dependence of carrier-mediated auxin uptake by suspension-cultured crown gall cells

1. The dependence on external hydrogen ion concentration of the carrier-mediated components of the uptakes of indol-3-yl acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4D) by suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch. is examined. The initiall rates of uptake of IAA and 2,4 D from 0.30 mol/l solutions exhibit pH optima of approximately pH 5.0 and pH 4.0 respectively.-2. The inherent difficulties, especially at low pH, of estimating the carrier-mediated uptake component in the presence of the large diffusive uptake of undissociated acid are discussed.-3. At pH 5.0, the uptakes of the two auxins are characterised by similar Michaelis Constants (0.65 and 0.80 mol/l) and Maximum Velocities (0.87 and 1.0 pmol/min/mg cells) for IAA and 2,4D respectively.-4. A dependence of carrier-mediated auxin uptake on cations other than hydrogen ions could not be demonstrated. It is suggested that uptake of auxin anions may occur by co-transport with hydrogen ions.Abbreviations IAA indol-3-yl acetic acid - 2,4 D 2,4-dichlorophenoxyacetic acid