Kinetics of nucleoside transport in human erythrocytes. Alterations during blood preservation

The transmembrane equilibration of radiolabeled uridine was measured by rapid kinetic techniques in human erythrocytes from freshly drawn blood and in the same cells during conventional storage of the blood as well as in cells from outdated blood. Our results confirm earlier reports that the maximum velocity of uridine equilibrium exchange (Vee) at 25 degrees C is about 30% lower in outdated than fresh red cells, whereas the opposite is the case for the Michaelis-Menten constant for equilibrium exchange (Kee), and that maximum zero-trans efflux (Vzt21) is about 4-times greater than maximum zero-trans influx (Vzt12) in outdated cells (directional asymmetry), whereas they are about the same in fresh red cells. At 25 degrees C, the nucleoside-loaded carrier of fresh cells moves on the average 6-times more rapidly than the empty carrier, whereas the differential mobility of loaded and empty carrier from outdated cells is about 15-fold. Our results also show that greater efflux than influx in outdated cells is not due to a general leakiness of outdated cells, that the differences in kinetic properties of the transporter developed during the first two weeks of blood storage and that the differences are greatly amplified when transport is measured at 5 degrees C rather than 25 degrees C. At 5 degrees C, the loaded carrier from outdated red cells moves about 325-times more rapidly than the empty carrier and maximum zero-trans efflux exceeds maximum zero-trans influx about 14-times, whereas the transport of fresh cells exhibits directional symmetry just as at 25 degrees C. The changes in kinetic properties of transport induced by temperature and storage are probably related to structural alterations in the plasma membrane and suggest that the operation of carrier is subject to modification by the membrane environment. Other results show that the kinetics of the sugar transport of human red cells is not affected in the same manner by blood storage as those of the nucleoside transporter.     

The transport of chloroquine across human erythrocyte membranes is mediated by a simple symmetric carrier

The kinetic properties of the mediated transport of chloroquine in human erythrocytes are investigated. The high rates of translocation across the cell membrane and high adsorbance properties to glass surfaces have led to the development of new techniques for measuring initial rates of transport. Three different methodological procedures are used to accomplish a complete kinetic characterization of the system. All measurements were done at 25°C. Under zero-trans conditions the system displays complete symmetry, the Michaelis constants being 39.2±2.4 μM for influx and 36.6±5.6 μM for efflux. The respective maximal velocities are 206.4±36.0 μM·min^?1 and 190.0±7.8 μM·min^?1. Under equilibrium-exchange conditions the Michaelis constant is 108.6±15.6 μM and the maximal velocity is 630.3±50.4 μM·min^?1. This 3-fold increase in both K and V over the zero-trans values indicates that the rate-limiting step in the transport of chloroquine is the movement of the unloaded carrier. The kinetic data are consistent with the prediction of a simple carrier model.     

Effects of temperature on the transport of nucleosides in guinea pig erythrocytes

The initial rate of [14C]uridine transport by guinea pig erythrocytes was investigated at different temperatures. At 37, 22, and 10 degrees C the concentration dependence of uridine zero-trans influx and equilibrium exchange influx was resolved into two components; (a) a saturable component which followed simple Michaelis-Menten kinetics and which was inhibited by nitrobenzylthioinosine, and (b) a linear component of low magnitude and insensitive to nitrobenzylthioinosine inhibition. The maximum velocity, Vmax, of zero-trans uridine influx for the saturable transport system was 70-fold higher at 37 than 10 degrees C (1.24, 0.20, and 0.018 mmol/L of cells per hour at 37, 22, and 10 degrees C, respectively). Similarly, the apparent affinity, Km, for zero-trans influx decreased as the temperature was lowered (0.27, 0.066, and 0.038 mM at 37, 22, and 10 degrees C, respectively). In contrast, uridine equilibrium exchange influx was less temperature dependent (Vmax, 2.80, 0.89, and 0.14 mmol/L of cells per hour; apparent Km 0.61, 0.36, and 0.24 mM at 37, 22, and 10 degrees C, respectively). These results demonstrate that the mobility of the empty carrier is impaired to a greater extent than the mobility of the loaded carrier temperature decreased. However, the kinetic constants for zero-trans uridine influx and efflux at 37 degrees C were similar, indicating that the nucleoside transporter exhibited directional symmetry at 37 degrees C. Arrhenius plots of the maximum velocity for equilibrium exchange and zero-trans uridine influx were discontinuous above 25 degrees C, but between 20 and 5 degrees C the plots were linear (Ea = 22 and 30 kcal/mol for equilibrium exchange and zero-trans influx, respectively.     

Adenine and hypoxanthine transport in human erythrocytes: distinct substrate effects on carrier mobility.

Transport of adenine and hypoxanthine in human erythrocytes proceeds via two mechanisms: (1) a common carrier for both nucleobases and (2) unsaturable permeation 4-5-fold faster for adenine for hypoxanthine. The latter process was resistant to inactivation by diazotized sulfanilic acid. Carrier mediated transport of both substrates was investigated using zero-trans and equilibrium exchange protocols. Adenine displayed a much higher affinity for the carrier (Km approximately 5-8 microM) than hypoxanthine (Km approximately 90-120 microM) but maximum fluxes at 25 degrees C were generally 5-10-fold lower for adenine (Vmax approximately 0.6-1.4 pmol/microliters per s) than for hypoxanthine (Vmax approximately 9-11 pmol/microliters per s). The carrier behaved symmetrically with respect to influx and efflux for both substrates. Adenine, but not hypoxanthine reduced carrier mobility more than 10-fold. The mobility of the unloaded carrier, calculated from the kinetic data of either hypoxanthine or adenine transport, was the same thus providing further evidence that these substrates share a common transporter and that their membrane transport is adequately described by the alternating conformation model of carrier-mediated transport.     

Kinetics of glucose transport in human erythrocytes: zero-trans efflux and infinite-trans efflux at 0 degree C

The kinetic features of glucose transport in human erythrocytes have been the subject of many studies, but no model is consistent with both the kinetic observations and the characteristics of the purified transporter. In order to reevaluate some of the kinetic features, initial rate measurements were performed at 0 degree C. The following kinetic parameters were obtained for fresh blood: zero-trans efflux Km = 3.4 mM, Vmax = 5.5 mM/min; infinite-trans efflux Km = 8.7 mM, Vmax = 28 mM/min. For outdated blood, somewhat different parameters were obtained: zero-trans efflux Km = 2.7 mM, Vmax = 2.4 mM/min; infinite-trans efflux Km = 19 mM, Vmax = 23 mM/min. The Km values for fresh blood differ from the previously reported values of 16 mM and 3.4 mM for zero-trans and infinite-trans efflux, respectively (Baker, G.F. and Naftalin, R.J. (1979) Biochim. Biophys. Acta 550, 474-484). The use of 50 mM galactose rather than 100 mM glucose as the infinite-trans sugar produced no change in the infinite-trans efflux Km values but somewhat lower Vmax values. Simulations indicate that initial rates were closely approximated by the experimental conditions. The observed time courses of efflux are inconsistent with a model involving rate-limiting dissociation of glucose from hemoglobin (Naftalin, R.J., Smith, P.M. and Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235-249). The results presented here support the adequacy of the carrier model to account for the kinetics.     

Substrate-specific differences in the rate of bile acid carrier reorientation: studies on human placental basal vesicles.

The initial rate of transport of the bile acid glycocholic acid (GCA) has been measured in influx and efflux across placental basal membrane vesicles, and the mechanism of inhibition of its transport by the analogue taurochenodeoxycholic acid (TCDCA) analysed kinetically. This analogue, although trans-stimulating GCA efflux, inhibits influx in a way which does not depend upon substrate concentration; moreover, its potency as an inhibitor is markedly influenced by whether it is placed on one or on both sides of the vesicles membrane. These findings can be accounted for by postulating that both GCA and TCDCA are translocated through the carrier, but that the rate of loaded carrier reorientation is higher than that of the free carrier only when loaded with TCDCA and not with GCA.     

The predicted ATP-binding domains in the hexose transporter GLUT1 critically affect transporter activity.

The glucose transporter GLUT1 has three short amino acid sequences (domains I-III) with homology to typical ATP-binding domains. GLUT1 is a facilitative transporter, however, and transports its substrates down a concentration gradient without a specific requirement for energy or hydrolysis of ATP. Therefore, we assessed the functional role of the predicted ATP-binding domains in GLUT1 by site-directed mutagenesis and expression in Xenopus oocytes. For each mutant, we determined the level of protein expression and the kinetics of transport under zero-trans influx, zero-trans efflux, and equilibrium exchange conditions. Although all five mutants were expressed at levels similar to that of the wild-type GLUT1, each single amino acid change in domains I or III profoundly affected GLUT1 function. The mutants Gly116-->Ala in domain I and Gly332-->Ala in domain III exhibited only 10-20% of the transport activity of the wild-type GLUT1. The mutants Gly111-->Ala in domain I and Leu336-->Ala in domain III showed altered kinetic properties; neither the apparent Km nor the Vmax for 3-methylglucose transport were increased under equilibrium exchange conditions, and they did not show the expected level of countertransport acceleration. The mutant Lys117-->Arg in domain I showed a marked increase in the apparent Km for 3-methylglucose transport under zero-trans efflux and equilibrium exchange conditions while maintaining countertransport acceleration. These results indicate that the predicted ATP-binding domains I and III in GLUT1 are important components of the region in GLUT1 involved in transport of the substrate and that their integrity is critical for maintaining the activity and kinetic properties of the transporter.     

Mechanism of proline transport in Escherichia coli K12. I. Effect of a membrane potential on the kinetics of 2H+/proline symport in cytoplasmic membrane vesicles

The stoichiometric coupling mechanism of the membrane potential (delta psi) in the reaction of H+/proline symport was investigated kinetically, using cytoplasmic membrane vesicles of the proline carrier-overproducing strain of Escherichia coli MinS/ pLC4 -45. When a delta psi was imposed across the cytoplasmic membrane by respiration, the Michaelis constant of transport (Kt) was lowered to about 1 microM, which was 2 orders of magnitude smaller than that of passive influx and efflux, and the maximum velocity (Vmax) was concomitantly enhanced as an exponential function of delta psi. Thermodynamically, the carrier translocated proline with a stoichiometry of 2 mol of protons versus 1 mol of substrate when driven by a delta psi at pH 8.0. Data on the delta psi dependence of Vmax of proline transport could be explained quantitatively by the Geck-Heinz hypothesis (Geck, P., and Heinz, E. (1976) Biochim, Biophys. Acta 443, 49-63). A symmetrical model of the 2H+/proline symport via formation of a carrier/H+/substrate (CH+H+S) intermediate is proposed. In this model, the effect of delta psi on the Kt was resolved as stimulation of formation of a transport intermediate, whereas the effect of delta psi on the Vmax was explained by enhancement of translocation of loaded carriers between the two sides of the membrane.     

Kinetic asymmetry of renal Na+-L-lactate cotransport. Characteristic parameters and evidence for a ping pong mechanism of the trans-stimulating exchange by pyruvate

Brush border vesicles prepared from horse renal cortex were used to study the kinetic properties of the Na+-L-lactate carrier on the outer and inner faces of the membrane. Two methods were applied for these measurements (in the absence of an electrical gradient): a direct method using influx and efflux kinetics, and an indirect method applied to trans-stimulated influx kinetics using membrane vesicles preloaded with various pyruvate concentrations (the latter enabled us to observe simultaneously the inner and outer carrier properties). Kinetic parameters obtained by the first method have shown that under sodium lactate chemical gradient, the carrier efficiency (estimated by the ratio of k = Vm/Km) is higher for the influx than efflux, a mechanism indicating a kinetic asymmetry of the transport. This difference remains at chemical equilibrium of solute concentration. The similarity of outer and inner affinity of sodium permits one to conclude that the kinetic asymmetry of the sodium lactate transport is related to the lactate-carrier interaction and not to that of the sodium-carrier. The second method using the pyruvate trans-activation effect (under sodium chemical equilibrium) has shown an affinity of lactate (Kt(out) = 1.1 mM), about 15 times higher for the carrier in the extracellular orientation than that of pyruvate for the carrier in the intracellular orientation (Kt(pyr) = 36 mM). This method has demonstrated a ping pong mechanism for the trans-activation exchange which accounts for a selective pore carrier model like a gated channel. These asymmetric properties are related to the AS glide sequential model (A and S being Na+ and lactate, respectively) proposed previously for the Na-L-lactate cotransport and to a different accessibility of the organic solute but not of the sodium on the two membrane faces.     

Physiological Characterization of Root Zn2+ Absorption and Translocation to Shoots in Zn Hyperaccumulator and Nonaccumulator Species of Thlaspi

Radiotracer techniques were employed to characterize 65Zn2+ influx into the root symplasm and translocation to the shoot in Thlaspi caerulescens, a Zn hyperaccumulator, and Thlaspi arvense, a nonaccumulator. A protocol was developed that allowed us to quantify unidirectional 65Zn2+ influx across the root-cell plasma membrane (20 min of radioactive uptake followed by 15 min of desorption in a 100 [mu]M ZnCl2 + 5 mM CaCl2 solution). Concentration-dependent Zn2+ influx in both Thlaspi species yielded nonsaturating kinetic curves that could be resolved into linear and saturable components. The linear kinetic component was shown to be cell-wall-bound Zn2+ remaining in the root after desorption, and the saturable component was due to Zn2+ influx across the root-cell plasma membrane. This saturable component followed Michaelis-Menten kinetics, with similar apparent Michaelis constant values for T. caerulescens and T. arvense (8 and 6 [mu]M, respectively). However, the maximum initial velocity for Zn2+ influx in T. caerulescens root cells was 4.5-fold higher than for T. arvense, indicating that enhanced absorption into the root is one of the mechanisms involved in Zn hyperaccumulation. After 96 h 10-fold more 65Zn was translocated to the shoot of T. caerulescens compared with T. arvense. This indicates that transport sites other than entry into the root symplasm are also stimulated in T. caerulescens. We suggest that although increased root Zn2+ influx is a significant component, transport across the plasma membrane and tonoplast of leaf cells must also be critical sites for Zn hyperaccumulation in T. caerulescens.     

Modeling Facilitative Sugar Transporters: Transitions Between Single and Double Ligand Occupancy of Multiconformational Channel Models Explain Anomalous Kinetics

The four-state simple carrier model (SCM) is employed to describe ligand translocation by diverse passive membrane transporters. However, its application to systems like facilitative sugar transporters (GLUTs) is controversial: unidirectional fluxes under zero-trans and equilibrium-exchange experimental conditions fit a SCM, but flux data from infinite-cis and infinite-trans experiments appear not to fit the same SCM. More complex kinetic models have been proposed to explain this ``anomalous' behavior of GLUTs, but none of them accounts for all the experimental findings. We propose an alternative model in which GLUTs are channels subject to conformational transitions, and further assume that the results from zero-trans and equilibrium-exchange experiments as well as trans-effects corresponds to a single-occupancy channel regime, whereas the results from the infinite-cis and infinite-trans experiments correspond to a regime including higher channel occupancies. We test the plausibility of this hypothesis by studying a kinetic model of a two-site channel with two conformational states. In each state, the channel can bind the ligand from only one of the compartments. Under single-occupancy, for conditions corresponding to zero-trans and equilibrium-exchange experiments, the model behaves as a SCM capable of exhibiting trans-stimulations. For a regime including higher degrees of occupancy and infinite-cis and infinite-trans conditions, the same channel model can exhibit a behavior qualitatively similar to a SCM, albeit with kinetic parameters different from those for the single-occupancy regime. Numerical results obtained with our model are consistent with available experimental data on facilitative glucose transport across erythrocyte membranes. Hence, if GLUTs are multiconformational channels, their particular kinetic properties can result from transitions between single and double channel occupancies. Received: 12 April 1995/Revised: 28 August 1995     

Na- and Cl-dependent glycine transport in human red blood cells and ghosts. A study of the binding of substrates to the outward-facing carrier

Na- and Cl-dependent glycine transport was investigated in human red blood cells. The effects of the carrier substrates (Na, Cl, and glycine) on the glycine transport kinetics were studied with the goal of learning more about the mechanism of transport. The K1/2-gly was 100 microM and the Vmax-gly was 109 mumol/kg Hb.h. When cis Na was lowered (50 mM) the K1/2-gly increased and the Vmax-gly decreased, which was consistent with a preferred order of rapid equilibrium loading of glycine before Na. Na-dependent glycine influx as a function of Na concentration was sigmoidal, and direct measurement of glycine and Na uptake indicated a stoichiometry of 2 Na:1 glycine transported. The sigmoidal response of glycine influx to Na concentration was best fit by a model with ordered binding of Na, the first Na with a high K1/2 (greater than 250 mM), and the second Na with a low K1/2 (less than 10.3 mM). In the presence of low Cl (cis and trans 5 mM), the K1/2-gly increased and the Vmax-gly increased. The Cl dependence displayed Michaelis-Menten kinetics with a K1/2-Cl of 9.5 mM. At low Cl (5 mM Cl balanced with NO3), the glycine influx as a function of Na showed the same stoichiometry and Vmax-Na but a decreased affinity of the carrier for Na. These data suggested that Cl binds to the carrier before Na. Experiments comparing influx and efflux rates of transport using red blood cell ghosts indicated a functional asymmetry of the transporter. Under the same gradient conditions, Na- and Cl-dependent glycine transport functioned in both directions across the membrane but rates of efflux were 50% greater than rates of influx. In addition, the presence of trans substrates modified influx and efflux differently. Trans glycine largely inhibited glycine efflux in the absence or presence of trans Na; trans Na largely inhibited glycine influx and this inhibition was partially reversed when trans glycine was also present. A model for the binding of these substrates to the outward-facing carrier is presented.     

Kinetic analysis of L-lactate transport in human erythrocytes via the monocarboxylate-specific carrier system

Three parallel pathways of L-lactate transport across the membrane of human red blood cells can be discriminated: (a) by nonionic diffusion; (b) via the band 3 anion exchange protein; and (c) via a specific monocarboxylate carrier system. Influx of lactate via the latter system leads to alkalinization of the medium, suggesting lactate-proton symport. Kinetic analysis of initial lactate influx via the monocarboxylate carrier indicates a symport system with ordered binding of the two ligands, in the sense that a proton binds first to the translocator, followed by lactate binding to the protonated carrier. The influence of varying trans-pH under conditions of net (zero-trans) flux with constant cis-pH indicates that the monocarboxylate translocator should be considered as a mobile carrier, with the ligand-binding sites exposed alternatively to the outside and the inside of the membrane.     

Kinetic analysis of l-lactate transport in human erythrocytes via the monocarboxylate-specific carrier system

Three parallel pathways of l-lactate transport across the membrane of human red blood cells can be discriminated: (a) by nonionic diffusion; (b) via the band 3 anion exchange protein; and (c) via a specific monocarboxylate carrier system. Influx of lactate via the latter system leads to alkalinization of the medium, suggesting lactate-proton symport. Kinetic analysis of initial lactate influx via the monocarboxylate carrier indicates a symport system with ordered binding of the two ligands, in the sense that a proton binds first to the translocator, followed by lactate binding to the protonated carrier. The influence of varying trans-pH under conditions of net (zero-trans) flux with constant cis-pH indicates that the monocarboxylate translocator should be considered as a mobile carrier, with the ligand-binding sites exposed alternately to the outside and the inside of the membrane.     

The asymmetric effect of lanthanides on Na+-gradient-dependent Ca2+ transport in synaptic plasma membrane vesicles

Lanthanides (La3+, Pr3+ and Tb3+) inhibit Na+-gradient-dependent Ca2+ influx into synaptic plasma membrane vesicles. 50% inhibition is obtained by 7 microM lanthanide concentration. The inhibition of the Na+-gradient-dependent Ca2+ uptake exhibits competitive kinetic behaviour. The apparent Km of the Ca2+ influx is increased from 50 microM in the absence of lanthanides to 118 microM in the presence of La3+, 170 microM in the presence of Pr3+ and 130 microM in the presence of Tb3+. The maximal reaction velocity is not altered (8.35 nmol Ca2+ transported per mg protein per min in the absence of lanthanides and 8.16 nmol/mg per min in the presence of lanthanides). Lanthanides also inhibited Na+-gradient-dependent Ca2+ efflux from synaptic plasma membrane vesicles that were preloaded with Ca2+ in a Na+-gradient-dependent manner. Introduction of La3+ into the interior of the synaptic plasma membrane vesicles by rapid freezing of the vesicles in liquid N2 and slow thawing had no effect on either Na+-gradient-dependent Ca2+ influx or efflux. Synaptic plasma membrane vesicles can be preloaded with Ca2+ also in an ATP-dependent manner. This form of Ca2+ uptake is also inhibited by La3+ though at higher concentrations than the Na+-gradient-dependent Ca2+ uptake. Na+-gradient-dependent efflux from synaptic plasma membrane vesicles preloaded in an ATP-dependent fashion ('inside-out' vesicles) unlike efflux from synaptic plasma membrane vesicles preloaded in a Na+-gradient-dependent manner was not inhibited by La3+. These findings suggest that the inhibition by La3+ is manifested asymmetrically on both sides of the synaptic plasma membrane. Lanthanides are probably not transported via the Na+-Ca2+ exchanger since Tb3+ entry measured by fluorescence of Tb3+-dipicolinic acid complex formation occurred at high Tb3+ concentrations only (1.5 mM or above) and was not Na+-gradient dependent.     

Glucose transporter in bovine mammary epithelial cells is an asymmetric carrier that exhibits cooperativity and trans-stimulation

Glucose transport kinetics were quantified in isolated bovine mammary epithelial cells using 3-O-methyl-D-glucose. Isolated cells retained satisfactory viability and glucose uptake activity, which was inhibited by cytochalasin B, phloretin, HgCl2, and low temperature. Initial rates of entry were measured over a 15-s interval at 37 degrees C under zero-trans, equilibrium-exchange, high-cis, and high-trans concentrations of 3-O-methyl-D-glucose between 0 and 20 mM. The combined set of rate measurements from all experimental conditions was fit to the fixed-site carrier model by nonlinear regression to estimate parameters of transport. For the regression between predicted and observed initial rates, r2 was 0.97. Forward Vmax was estimated at 18.2 nmol.min-1.mg protein-1, and the Michaelis constant was 8.29 mM. The cooperativity parameter was 1.63, trans-stimulation was 2.13-fold, and asymmetry was 2.06-fold. On the basis of the kinetic parameters, variations in intracellular glucose concentrations are not responsible for the range of glucose uptakes by bovine mammary glands observed in vivo.     

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [¹⁴C]2,4-dichlorophenoxyacetic acid and [³H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [³H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [¹⁴C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turned-over proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999