Tests of the carrier model for ion transport by nonactin and trinactin.

The fluxes of K+ and NH+4 carried by nonactin and trinactin across thin lipid membranes have been measured as functions of ion activity, electric potential and time. In agreement with the predictions of a version of the carrier model in common use, the shape of the initial current-voltage relation is independent of the activity of the electrolyte, alpha-i, while the ratio of the initial conductance, G-o, to the steady-state conductance, G infinity, increases according to G-o/G infinity equals const1+const2 times alpha-i. For trinactin the data presented allow the estimation of the rate constants of the carrier process (in the limit of zero potential) in a manner which does not assume any particular variation with potential for the constants. Using empirically determined functions of potential, a complete set of values is also available for nonactin. The curve fitting which is necessary is described in the following paper. The data presently available for valinomycin are sufficient neither to test the model nor to determine a complete set of constants.

Steady-state ion transport by nonactin and trinactin.

The steady-state fluxes of Na+, K+, and NH4+ carried by nonactin and trinactin across thin lipid membranes have been measured as functions of ion activity, carrier concentration, and the applied potential. In agreement with earlier studies the conductance, G(O), is found to be proportional to the carrier concentration and, for low activities, to the ion activity. The determination of the dependence of G(O) on activity at high activities is, however, apparently obscured by changes in the concentration of carrier in the membrane. Using the values for the rate constants at zero potential which were determined in the preceding paper, it is possible to adjust the potential dependence of the constants so as to achieve a reasonable fit to the current-voltage relations. The data presented provide further evidence that a single molecule of nonactin or trinactin acts cyclicly as a carrier of univalent cations.

Ion transport in the simplest single file pore.

A kinetic scheme is developed to describe single-file transport through pores containing up to two ions which may be of different species. The solution for the fluxes in terms of rate constants for entry, exit, and transfer is derived without specific assumptions about symmetry or the voltage and activity dependence of the constants. For a symmetrical pore the relation between the slope conductance at zero applied potential and ion activity can have two distinct regions in which the conductance increases linearly. Zero current or reversal potentials depend on the absolute values of the activities as well as their ratios. The use of this theory to describe the cation fluxes through the pores formed by gramicidin A will be considered in a subsequent paper. Here the model is discussed for a number of more specific assumptions, most extensively the following combination: (1) while entry to a pore is less likely when the pore is already occupied at the opposite end, this entry is still rapid; (2) exit is much more rapid when the pore is occupied by two ions; and (3) transfer from one end to the other of a singly occupied pore is rapid. With these assumptions and for a range of concentrations over which the fluxes are proportional to ion activities, the model predicts a flux ratio exponent nearly equal to 2, blocking by impermeant ions, rectification due to blocking particles on one side only, relief of block by increase in the permeant ion concentration on the opposite side, and anomalous variations of the conductance and zero current potential with mole ratio when the total concentration of the two permeants is held constant.

An upper limit for the effect of low frequency magnetic fields on ATP-sensitive potassium channels.

Currents have been recorded for ATP-sensitive potassium channels in excised patches of membranes from an insulin secreting cell line, CRI-G1. The multi-channel records have been analyzed to reveal the single-channel conductance, the frequency and duration of bursts and the frequency of flickers (with periods between 0.5 and 5 ms). Control records in the absence of applied magnetic fields are similar to those reported by others. Patches have been exposed to parallel static and low frequency magnetic fields including a combination satisfying the 'cyclotron resonance' condition. The fields were applied for 30 s periods interleaved with 30 s controls. No significant differences in channel properties were observed between the control and field exposed periods. The largest change in position of the peak of the distribution of opening and closing transitions was 3%.

Transient currents carried by the uncoupler, carbonyl cyanide m-chlorophenylhydrazone.

The weak acid uncoupler, carbonyl cyanide m-chlorophenylhydrazone, carries protons across lipid membranes. As predicted by the carrier model, at low pH, the current changes immediately following a jump in applied potential and then remains constant. By contrast at high pH, the currents relax from an initial value to a lower value as the carrier anions redistribute in the membrane. These relaxations are slower than those seen with other lipid-soluble anions which presumably explains why they had not been detected previously.

Ion movements in gramicidin pores. An example of single-file transport.

Experimental results on ion movement through gramicidin membrane channels are presented and discussed in terms of ion transport in the simplest single-file pore (for review see Urban, B.W. and Hladky, S.B. (1979) Biochim. Biophys. Acta 554, 410-429). Single-channel conductance and bi-ionic potential data for Na+, K+, Cs+, NH4+ and Tl+ are used to assign values to the rate constants of the model. Not all of the rate constants can be determined uniquely and simplifications are introduced to reduce the number of free parameters. The simplified model gives good quantitative fits to the experimental results for Na+, K+, Cs+ and NH4+. For Tl+, although the model accounts qualitatively for the salient features of the results, the quantitative agreement is less satisfactory. Predictions calculated from the model and the fitted rate constants are compared with independent data from blocking and tracer-flux measurements. In agreement with experiment, the model shows that only Tl+ blocks the Na+ conductance significantly. Furthermore, the exponent, n, in the tracer flux ratio rises, as observed, well above unity. The values for the rate constants suggest internal consistency of the model in that entry is always slower to singly occupied pores than to empty pores while exit is always faster from doubly as compared to singly occupied pores. The agreement between model prediction and experimental results suggests that the main features of ion transport in the gramicidin channel arise from cation-cation interaction in a single-file pore.

Ion channel formation by duramycin.

The formation of ion channels by the nonadecapeptide antibiotic duramycin was examined using black lipid membranes and using the patch-clamp technique. In black lipid membranes made from glyceryl monooleate or a phosphatidylcholine/phosphatidylethanolamine mixture, duramycin induced complex fluctuations in membrane conductance, some step-like and some which were incapable of being resolved into discrete conductance states. Both conductance and largest step size increased with time. A similar time-dependent increase in conductance was seen in patch-clamp experiments with HCA-7 Colony 29 human colonic epithelial cell. The channels displayed weak anion selectivity and the smaller channels formed in patches from epithelial cells showed weak inward-rectification. Channel formation by duramycin was achieved at lower concentrations when the black lipid membrane was made with phospholipid rather than with glyceryl monooleate. Lower concentrations were effective in generating conductances in epithelial cells than in bilayers. It is concluded that duramycin forms ion channels in both artificial and biological membranes. Accumulation of duramycin and coalescence of initially small channels into larger ones is considered to be responsible for the recorded behaviour and to final disruption of membranes.

Hypotonicity-induced changes in anion permeability of cultured rat brain endothelial cells.

Iodide efflux, an index of anion permeability, has been monitored in cultured rat brain endothelial cells. Following hypotonicity-induced swelling, large, rapid increases in permeability occur, the extent of these increases depending on the degree of hypotonicity. Such large responses are not observed with rat aortic endothelial cells. Results of anion substitution experiments suggest that iodide efflux is via a chloride channel rather than an exchanger. The efflux increase is blocked by NPPB (100 microM) but not by DIDS or DPC at 100 microM. It is dependent on intracellular ATP but unaffected by removal of external calcium. Increasing internal calcium using A23187 does not produce a change in efflux, but depletion of calcium reduces or eliminates the response to hypotonicity. The response is reduced by pimozide (2-50 microM) that inhibits the actions of calmodulin and by pBPB (10 microM) that affects phospholipase A2 activity. It is eliminated by 5-lipoxygenase inhibitors (L-656,224 and ETH615, 10 microM) but is unaffected by cyclo-oxygenase inhibitors (indomethacin and piroxicam, 1-100 microM). It is blocked by some modulators of P-glycoprotein activity, e.g., verapamil (100 microM), tamoxifen (50 microM), and progesterone (100 microM) but not by others, e,g., forskolin (40 microM), dideoxyforskolin (40 microM), quinidine (100 microM) and cyclosporin A (10 microM).

Influences of glutathione on anionic substrate efflux in tumour cells expressing the multidrug resistance-associated protein, MRP1.

The ATP-dependent transport of natural product drugs, e.g. vincristine, by multidrug resistance-associated protein (MRP1) requires reduced glutathione (GSH), whilst that of anionic substrates does not. The present results suggest, however, that GSH can modulate transport of anionic species. Efflux of fluorescent anionic substrates was measured from adherent MRP1-expressing human multidrug-resistant lung tumour cells, COR-L23/R, and drug-sensitive parental cells. As expected, much greater efflux of calcein, methylfluorescein-glutathione (GS-MF), and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was observed from the resistant cells. Unexpectedly, lowering GSH levels in COR-L23/R cells by inhibiting GSH synthesis with buthionine sulfoximine decreased efflux of calcein and of GS-MF (3-fold and 1.6-fold) but not efflux of BCECF. Transport of the anionic conjugate dinitrophenyl-glutathione ([(3)H]DNP-SG) was investigated by following its uptake into inside-out plasma membrane vesicles prepared from the MRP1-expressing cells. At least 90% of the ATP-dependent uptake was blockable by the anti-MRP1 antibody QCRL-3 and 100 microM vincristine inhibited uptake but only in the presence of 1--3 mM GSH, suggesting MRP1 to be the protein primarily responsible for this transport. Agents shown to reduce efflux of calcein from resistant cells, i.e. indomethacin, MK-571, and probenecid, also inhibited [(3)H]DNP-SG uptakes, consistent with MRP1 being responsible for export of calcein. At concentrations achievable within cells, GSSG (70 microM) inhibited uptake whereas GSH (1 and 3 mM) enhanced uptake. We suggest that variations in both GSH and GSSG levels within cells may affect MRP1-mediated anion transport.

Can we use rate constants and state models to describe ion transport through gramicidin channels?

Can we use rate constants and state models to describe ion transport through gramicidin channels? Maybe, but only if rate constants are just proportionality constants between rates and probabilities of observing states of the channel. This approach is natural if the system of channel plus ions (plus water) is almost always in one or another of a small number of identifiable states. Many features of ion transport through gramicidin, including the conductance-concentration relationship, concentration-dependent permeability ratios, anomalous mole fraction effect and to some extent flux ratio exponents, are consistent with a description in which there are four occupation 'states' of the pore: only water; an ion at one end; an ion at the other; and ions at both ends. Current-voltage relationships can (and must) also be fitted, but until there is a theory to predict the potential dependence of the rate constants this success will remain hollow. Other features have resisted interpretation. These include the failures to determine 'binding constants' consistent with all the data; the variation of flux ratio exponents with ion type; and, probably, the variation of the currents with asymmetrical ion concentrations. Nevertheless, state models still have one attractive feature, they allow consideration of the effects that one ion within the pore has on the movements of another.

Use of membrane vesicles to investigate drug interactions with transporter proteins, P-glycoprotein and multidrug resistance-associated protein.

BACKGROUND: The ATP-dependent drug transporter proteins, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) are known to be involved in drug efflux that reduces drug accumulation and so renders tumor cells resistant to the cytotoxic effects of a number of anticancer agents. The ways in which these transporters bring about drug expulsion are not fully explained and may involve intracellular factors as well. Thus detailed evidence may be difficult to obtain from studies on intact cells. MATERIAL AND METHODS: Inside-out plasma membrane vesicles prepared from multidrug-resistant cells expressing high amounts of Pgp or of MRP provide a simpler system for investigating the interactions of putative substrates and resistance modifiers with the transport process. We consider here some aspects of the accumulation of radiolabelled vincristine and of dinitrophenol glutathione conjugate by these vesicles and demonstrate the usefulness of this approach for determining whether potential inhibitors have their effects on transport at the cell membrane or by more indirect means. CONCLUSIONS: We show that information gained from analysis of the ATP-dependence, time course and osmotic sensitivity of accumulation is helpful in distinguishing between transport and changes in binding. We have also used the technique to demonstrate the effects of the resistance modifier, XR-9051 on Pgp-mediated transport and to explore interactions of MK571, indomethacin and ethacrynic acid with MRP.

The effect of diffusion and convection on the rate of transfer of solutes across an interface.

The transfer of substances across the interface between water and a membrane or between water and a solvent occurs in series with transport up to and away from the interface. These processes have been difficult to resolve. Recently D. M. Miller (Biochim Biophys Acta 856: 27-35, 1986) has used a moving drop technique to measure the rates of transfer of short-chain alcohols and tritiated water between water and n-octanol. This technique produces equivalent unstirred layers which are less than about 10 microns thick. Based on the trends in the observed rates of phase transfer, he proposes that the transfer is limited by the actual interfacial step. If so, water-oil interfacial transfer would be sufficiently slow to limit the rate of permeation of lipid membranes by these substances. It is shown here that the observed rates of phase transfer can be explained quantitatively if they are limited by convection or by diffusion across the combination of 5-10 microns unstirred layers both inside and outside the moving drops. For water, comparison of the observed rates with the rate of evaporation from a clean surface, suggests that the interfacial step at the water-octanol interface is not rate-limiting.

Ion transport and displacement currents with membrane-bound carriers: the theory for voltage-clamp currents, charge-pulse transients and admittance for symmetrical systems.

The standard carrier model for ion transport by a one-to-one mechanism is developed to predict the time-dependent currents for systems that are symmetrical at zero applied potential. The complete solution for ions and carriers bearing any charge is derived by assuming that the concentration of ions in the membrane is low and either that the applied potential is small or that the applied potential affects equally all of the association and dissociation reactions between the ions and the carriers. The response to an abruptly applied potential is then given by the sum of a constant and two declining exponential terms. The time constants of these relaxations are described by the equations derived for neutral carriers by Stark, Ketterer, Benz and Lüger in 1971 (Biophys. J. 11:981). The sum of the amplitudes of the exponentials for small applied potentials obeys a relation like that first derived by Markin and Liberman in 1973 (Biofizika 18:453). For small applied potentials expressions are also provided for the voltage transients in charge-pulse experiments and for the membrane admittance.

Ion currents through pores. The roles of diffusion and external access steps in determining the currents through narrow pores.

External access steps, which may include restricted aqueous diffusion, are introduced into a kinetic model for ion transport through narrow pores. The conductance-concentration relation and the concentration dependence of the biionic permeability are calculated using two alternative assumptions: (a) access to the mouth of the pore is allowed only when no ion is within the lumen or at either mouth; (b) ions remain at the mouth only very transiently. With either assumption the concentration dependence of the fluxes is the same as in previous treatments in which all steps in access were lumped into a single process. Also as before, the biionic permeability ratio is independent of concentration so long as the lumen is never doubly occupied. For narrow pores, such as those formed by gramicidin A, the slowest external portion of the access process must occur close to the pore's mouth, and thus the region an ion must occupy to gain access is small. As a consequence, the probability of finding an ion within this region is also small. On this basis, it is argued that the second assumption is appropriate for these pores. The kinetic equations that result are identical to those used by Urban, B., S.B. Hladky, and D.A. Haydon (1980, Biochim. Biophys. Acta. 602:331-354).

Potential difference and the distribution of ions across the human red blood cell membrane; a study of the mechanism by which the fluorescent cation, diS-C3-(5) reports membrane potential.

1. The mechanism by which the fluorescent, cationic dye diS-C3-(5) responds to the membrane potential of red blood cells has been investigated. 2. The dye in aqueous solution absorbs most strongly at 650 nm. Addition of white, haemoglobin-free membranes red shifts the absorption maximum ca. 20 nm, while addition of membrane-free cell lysate results in the appearance of a new dye absorption peak at 590 nm. Thus the dye binds both to cell membranes and to cell contents. The component of the cytoplasm which binds the dye is non-dialysable, presumably haemoglobin. 3. Dye added to a suspension of intact cells shows a strong absorption at 590 nm indicating that the dye has bound to the cell contents and that the membrane is permeable to the dye. 4. The amount of dye which partitions into (and on to) the cells can be determined, as reported by Sims, Waggoner, Wang & Hoffman (1974), from the fluorescence of the dye remaining in the supernatant after the cells are centrifuged to the bottom of the suspension. In most conditions the proportion of the cell associated dye which is either free inside the cell or bound to the outside face of the membrane is negligible compared to the proportion bound to the cell contents. 5. On the assumption that the dye is not actively transported, the ratio of the equilibrium dye activities inside and outside the cell, ai/ao, is determined by the membrane potential according to the Nernst relation. Driving the membrane potenial negative then increases the cell associated dye by increasing the activity ratio and hence ai and the amount of dye bound to cell contents. 6. At the known Donnan equilibrium potential the internal dye activity can be calculated from the external activity. An empirical relation between cell associated dye and internal activity has been determined by measuring the dye partition between cells and medium at different external activities. 7. Using this empirial relation, and providing that any changes in cell composition do not affect the dye binding, the internal activity at any potential can be calculated from the measured amount of cell associated dye. The external activity can be estimated fluorimetrically. The membrane potential is then calculated from the activity ratio. 8. The membrane potenial of cells has been altered by adding valinomycin in the presence of different K gradients. Under the conditions used, the 'constant field' permeability for K-Val is 15-20 times that of Cl. 9. Dye binding to haemoglobin is influenced by pH and thus dye partitioning into cells changes with intracellular pH. Increasing intracellular pH increases the amount of dye partitioned, while decreasing pH decreases this amount. 10. When large potentials are produced with valinomycin there is no change in intracellular pH. This result indicates that in red blood cells intracellular pH is determined by the external pH and the Cl concentration ratio and not by the membrane potentials. 11. DiS-C3-(5) can be used to estimate potentials across resealed ghost membranes...

Absence of effects of low-frequency, low-amplitude magnetic fields on the properties of gramicidin A channels.

The effects of static and low-frequency magnetic fields on gramicidin A channels have been investigated using bilayer patch clamp recording and a bridge technique capable of detecting 0.3% changes in the conductance of glyceryl monooleate membranes containing many channels. In the bridge technique the conductance was assessed using 10-ms voltage pulses applied at 10 Hz. Measurements were made for LiCl, KCl, and CsCl using magnetic fields of 50, 100, 500, and 5000 microT with the frequency scanned from 10-200 Hz. The combinations of static and low-frequency fields employed include the "cyclotron resonance" conditions at which effects had been predicted to occur. In no case was there any detectable change in conductance when the magnetic fields were applied or changed. Potassium currents through single gramicidin channels have been recorded for patches in which several channels may be open at once. Fields were applied for 2 min periods interleaved with 2 min controls. Methods have been developed to analyze the multichannel records to reveal the amplitude and duration of the channels together with the frequency, depth, and apparent period of flickers. No significant differences were observed between the control and field-exposed recording periods. The peak of the distribution of opening and closing transitions always coincided for fields on and off within the resolution, 0.4%, of the recordings. There are at least two types of flicker, one with typical period less than 0.1 ms, the other with typical period from 0.3-0.8 ms. Most of the latter were not complete closures with the conductance during a flicker 15-20% above the level for a full closure.