Testing the hypothesis that system y(+)L accounts for high- and low-transport phenotypes in chicken erythrocytes using L-leucine as substrate.

Experiments were carried out to test the hypothesis that system y(+)L accounts for the high (HT) and low (LT) amino-acid transport phenotypes in chicken erythrocytes and to explain the different effect of selective breeding on lysine and leucine fluxes. L: -Leucine transport was characterized in individuals which had been separated into two groups (HT and LT) according to their capacity to transport L: -lysine across the erythrocyte membrane. Whereas lysine influx (1 muM: ) in the two groups differed by 32-fold (HT/LT), leucine influx was not significantly different. Average rates (nmol/ L cells/ min) were: 227 (HT) and 7.0 (LT) for L: -lysine, and 8.9 (HT) and 7.1 (LT) for L: -leucine. The differential ability of L: -lysine and L: -leucine fluxes to discriminate between the HT and LT phenotypes was shown to be consistent with the interactions of these substrates with system y(+)L and to vary depending on the conditions of the assay. It is shown that the two phenotypes can be clearly discriminated by measuring L: -leucine influx in the presence of Li(+). These results support the hypothesis that the HT and LT phenotypes reflect alterations in the function of system y(+)L and illustrate that the choice of the appropriate substrate and medium composition must be carefully considered when investigating the consequences of either experimental or natural alterations of broad-scope transporters.

Epithelial cells isolated from chicken jejunum: an experimental model for the study of the functional properties of amino acid transport system b(0,+).

The transport of lysine has been investigated in epithelial cells isolated from chicken jejunum. The kinetics of lysine transport and the pattern of interaction with zwitterionic amino acids were consistent with system b(0,+) activity, the broad-spectrum and Na(+)-independent amino acid transporter. The half-saturation constant for lysine entry (K(m)+/-S.E.) was 0.029+/-0.002 mM and the flux was not affected significantly by Na(+) replacement with choline. Lysine influx was inhibited by L-leucine both in Na(+) and choline medium with inhibition constants (K(i)+/-S.E.) 0.068+/-0.006 mM (in Na(+)) and 0.065+/-0.009 mM (in choline). Other inhibitory amino acids (K(i)+/-S.E.) were (mM): L-tyrosine (0.073+/-0.018), L-methionine (0.15+/-0.015), L-cystine (0.42+/-0.04), L-cysteine (1.1+/-0.07), L-isoleucine (1.1+/-0.09), L-glutamine (1.8+/-0.16) and L-valine (2.5+/-0.13). Lysine exit was trans-accelerated (approx. 20 fold) by 2 mM L-lysine and L-leucine. The flux was resistant to pretreatment of the cells with p-chloromercuriphenylsulfonate (0.2 mM), which is an inhibitor of system y(+)L, the broad-spectrum and cation-modulated transporter.

System y+L-like activities account for high and low amino-acid transport phenotypes in chicken erythrocytes.

The functional properties of the transport of lysine across the chicken erythrocyte membrane were investigated. The animal population studied (male Leghorn chickens, 6-14 weeks old) was found to consist of two groups presenting either low (LT, 19 individuals) or high transport rates (HT, 20 individuals). The rates of influx in the two groups, measured at a concentration of l-lysine of 1 microm, differed by a factor of 34. The transport activities observed in LT and HT erythrocytes were compatible with the general features of system y+L, but showed some differences in specificity. The transporter in the LT group was found to bind l-lysine, l-leucine, l-methionine and l-glutamine with high affinity, in the presence of sodium, as described for system y+L in human erythrocytes. The activity present in HT erythrocytes exhibited a much lower affinity for l-leucine, but was able to interact strongly with l-glutamine and l-methionine. The specificity pattern of the HT transporter, has not been described in other cell types. In other respects, the properties of the two systems were similar. Sodium replacement with potassium, drastically reduced the affinity for l-leucine, without affecting lysine transport. Both transporters function as tightly coupled exchangers, are inactivated by p-chloromercuribenzene sulfonate and resistant to N-ethylmaleimide. These findings explain previous results obtained in selective breeding experiments of chicken with high and low amino-acid transport activity.

Cationic amino acid transport through system y+L in erythrocytes of patients with lysinuric protein intolerance.

We test the hypothesis that lysinuric protein intolerance (LPI), a rare autosomal recessive defect of cationic amino acid transport, results from the absence of the recently described y+L amino acid transporter. We compare fluxes of lysine (1 microM) into erythrocytes of normal subjects with those of patients homozygous for the LPI mutation. No significant differences in fluxes through system y+L in normal or LPI cells were found, excluding the possibility that system y+L cannot be expressed in patients with LPI. Reasons for supposing that there may be tissue-specific processing of two recently described genes encoding the y+L transporter are discussed. Polymerase chain reaction measurement of expression of these two genes in an erythroleukemic cell line suggests that alternatively there may be an as-yet-unidentified additional member of this gene family.

Mammalian amino acid transport system y+ revisited: specificity and cation dependence of the interaction with neutral amino acids.

A reevaluation of the specificity of system y+, the classical transporter for cationic amino acids is presented. System y+ has been defined as a transporter for cationic amino acids that binds neutral amino acids with lower affinity in the presence of Na+. The discovery of other transporters for cationic amino has suggested that some properties, originally attributed to system y+, may relate to other transport systems. Uncertainty concerns mainly, the affinity for neutral amino acids and the cation dependence of this interaction. Neutral amino acids (13 analogues tested) were found to bind to system y+ in human erythrocytes with very low affinity. Inhibition constants (Kiy, mm) ranged between 14.2 mm and >400 mm, and the strength of interaction was similar in the presence of Na+, K+ or Li+ (145 mm). In choline medium, no interaction was detected up to 20 mm of the neutral amino acid. Guanidinium ion (5 mm, osmolarity maintained with choline) potentiated neutral amino acid binding; the effect was most important in the case of l-norvaline which aligned with guanidinium ion is equivalent to arginine. This suggests cooperative interaction at the substrate site. The specificity of system y+ was shown to be clearly distinct from that of system y+L, a cationic amino acid transporter that accepts neutral amino acids with high affinity in the presence of Na+ and which influenced the classical definition of system y+.

The amino acid transport system y+L/4F2hc is a heteromultimeric complex.

4F2hc is an almost ubiquitous transmembrane protein in mammalian cells; upon expression in Xenopus laevis oocytes, it induces amino acid transport with characteristics of system y+L. Indirect evidence fostered speculation that function requires the association of 4F2hc with another protein endogenous to oocytes and native tissues. We show that expression of system y+L-like amino acid transport activity by 4F2hc in oocytes is limited by an endogenous factor and that direct covalent modification of external cysteine residue(s) of an oocyte membrane protein blocks system y+L/4F2hc transport activity, based on the following. 1) Induction of system y+L-like activity saturates at very low doses of human 4F2hc cRNA (0.1 ng/oocyte). This saturation occurs with very low expression of 4F2hc at the oocyte surface, and further increased expression of the protein at the cell surface does not result in higher induction of system y+L-like activity. 2) Human 4F2hc contains only two cysteine residues (C109 and C330). We mutated these residues, singly and in combination, to serine (C109S; CS1, C330S; CS2 and C109S-C330S, Cys-less). Mutation CS2 had no effect on the expressed system y+L-like transport activity, whereas C109S-containing mutants (CS1 and Cys-less) retained only partial y+L-like transport activity (30 to 50% of wild type). 3) Hg2+, the organic mercury compounds pCMB, and the membrane-impermeant pCMBS almost completely inactivated system y+L-like induced by human 4F2hc wild type and all the mutants studied. This was reversed by ss-mercaptoethanol, indicating that external cysteine residue(s) are the target of this inactivation. 4) Sensitivity to Hg2+ inactivation is increased by pretreatment of oocytes with ss-mercaptoethanol or in the C109S-containing mutants (CS1 and Cys-less). The increased Hg2+ reactivity of C109S-containing mutants supports the possibility that C109 may be linked by a disulfide bond to the Hg2+-targeted cysteine residue of the associated protein. These results indicate that 4F2hc is intimately associated with a membrane oocyte protein for the expression of system y+L amino acid transport activity. To our knowledge, this is the first direct evidence for a heteromultimeric protein structure of an organic solute carrier in mammals.

System y+L: the broad scope and cation modulated amino acid transporter.

The properties are discussed of system y+L, a broad scope amino acid transporter which was first identified in human erythrocytes. System y+L exhibits two distinctive properties: (a) it can bind and translocate cationic and neutral amino acids, and (b) its specificity varies depending on the ionic composition of the medium. In Na+ medium, the half-saturation constant for L-lysine influx was 9.5 +/- 0.67 microM and the inhibition constant (Ki) for L-leucine was 10.7 +/- 0.72 microM. L-Leucine is the neutral amino acid that binds more powerfully, whereas smaller analogues, such as L-alanine and L-serine interact less strongly (the corresponding inhibition constants were Ki,Ala, 0.62 +/- 0.11 mM; Ki,Ser, 0.49 +/- 0.08 mM). In the presence of K+, the carrier functions as a cationic amino acid specific carrier, but Li+ is able to substitute for Na+ facilitating neutral amino acid binding. The effect of the inorganic cations is restricted to the recognition of neutral amino acids; translocation occurs at similar rates in the presence of Na+, K+ and Li+. The only structural feature that appears to impair translocation is bulkiness and substrates with half-saturation constants differing by more than 100-fold translocate at the same rate. This suggests that translocation is largely independent of the forces of interaction between the substrate and the carrier site. System y+L activity has been observed in Xenopus laevis oocytes injected with the cRNA for the heavy chain of the 4F2 human surface antigen. 4F2hc is an integral membrane protein with a single putative membrane-spanning domain and it remains to be clarified whether it is part of the transporter or an activator protein.

Transporters for cationic amino acids in animal cells: discovery, structure, and function.

The structure and function of the four cationic amino acid transporters identified in animal cells are discussed. The systems differ in specificity, cation dependence, and physiological role. One of them, system y+, is selective for cationic amino acids, whereas the others (B[0,+], b[0,+], and y+ L) also accept neutral amino acids. In recent years, cDNA clones related to these activities have been isolated. Thus two families of proteins have been identified: 1) CAT or cationic amino acid transporters and 2) BAT or broad-scope transport proteins. In the CAT family, three genes encode for four different isoforms [CAT-1, CAT-2A, CAT-2(B) and CAT-3]; these are approximately 70-kDa proteins with multiple transmembrane segments (12-14), and despite their structural similarity, they differ in tissue distribution, kinetics, and regulatory properties. System y+ is the expression of the activity of CAT transporters. The BAT family includes two isoforms (rBAT and 4F2hc); these are 59- to 78-kDa proteins with one to four membrane-spanning segments, and it has been proposed that these proteins act as transport regulators. The expression of rBAT and 4F2hc induces system b[0,+] and system y+ L activity in Xenopus laevis oocytes, respectively. The roles of these transporters in nutrition, endocrinology, nitric oxide biology, and immunology, as well as in the genetic diseases cystinuria and lysinuric protein intolerance, are reviewed. Experimental strategies, which can be used in the kinetic characterization of coexpressed transporters, are also discussed.

Changes in membrane and surface potential explain the opposite effects of low ionic strength on the two lysine transporters of human erythrocytes.

The sucrose-induced stimulation of lysine influx in human erythrocytes has been attributed to the removal of a competitive inhibition exerted by Na+ on system y+ (Young, J. D., Fincham, D. A., and Harvey, C. M. (1991) Biochim. Biophys. Acta 1070, 111-118). We have reexamined this phenomenon separating the contribution of the two cationic amino acid transporters present in these cells (system y+ and system y+L). NaCl replacement with sucrose increased influx through system y+L, but decreased influx through system y+. We conclude that 1) the inhibition of system y+ is a response to the membrane depolarization that results from chloride removal, and 2) the stimulation of system y+L is due to the enhancement of the negative surface potential. Consistently, lysine influx through system y+L (in sucrose medium) was reduced by Na+, K+, Li+, and choline (K0.5 = 25-34 mM), the effect reaching a maximum at 35-40% of the original flux. Divalent cations (Ca2+ and Mg2+) were also inhibitory, but lower concentrations were required (K0.5 1.1-1.8 mM). The finding that sucrose stimulates uptake through changes in the surface potential explains similar effects observed in other cells with various cationic substrates.

The binding specificity of amino acid transport system y+L in human erythrocytes is altered by monovalent cations.

System y+L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na+ replacement with K+ does not affect lysine transport, but markedly decreases the affinity of the transporter for L-leucine and L-glutamine. This observation suggests that the specificity of system y+L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various amino acids in the presence of Na+, K+, Li+ and guanidinium ion. In agreement with the prediction, the specificity of system y+L was altered by the monovalent cations. In the presence of Na+, L-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - L-norleucine) strengthened binding. In contrast, bulkiness at the level of the beta carbon was detrimental. In K+, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li+ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na+; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards beta carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was restricted to certain analogues (e.g., L-leucine, L-glutamine, L-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids. The results suggest that the monovalent cations stabilize different carrier conformations.

Transport of nitric oxide synthase inhibitors through cationic amino acid carriers in human erythrocytes.

The interaction of arginine analogues, which are known to inhibit nitric oxide synthase, with two cationic amino acid transporters of human erythrocytes (systems y+ and y+L) was studied. Arginine and relevant analogues [NG-monomethyl-L-arginine (L-NMMA); NG-monomethyl-D-arginine (D-NMMA) and NG-nitro-L-arginine (L-NOARG)] were found to inhibit labeled lysine influx into intact erythrocytes. As expected, the pattern of inhibition reflected the contribution of the two distinct transport systems. All analogues showed a higher affinity for system y+L than for system y+. The half-saturation (inhibition) constants estimated for systems y+ and y+L (+/- SEM) were (microM): L-arginine, 55.7 +/- 5.4 and 2.4 +/- 0.1; L-NMMA, 151 +/- 13 and 7.5 +/- 0.5; D-NMMA, 2660 +/- 404 and 269 +/- 25; L-NOARG, 9414 +/- 169 and 594 +/- 35. The transport properties of the analogues were investigated using an assay based on the trans-stimulation of lysine efflux. The addition of saturating concentrations of unlabeled analogues to the external medium stimulated efflux of labeled lysine through systems y+L and y+, showing that the analogues can enter the cell through these pathways.

Membrane potential dependence of the kinetics of cationic amino acid transport systems in human placenta.

1. Mediated influx of L-lysine into human placental brush-border membrane vesicles occurs through two systems, one of lower affinity but high capacity, the other of very high affinity but low capacity. These transporters have features characteristic of systems y+ (the classical system) and y+L (recently described in the erythrocyte), respectively. 2. In solutions containing sodium the entry of lysine through the high-affinity system (y+L) is inhibited by the neutral amino acids L-leucine, L-methionine and L-glutamine with comparable high affinity. The removal of sodium reduces the affinity but not the maximal extent of this inhibition. Leucine and methionine, but apparently not glutamine, inhibit lysine entry through system y+ with a much lower affinity. 3. The influx of lysine through system y+ changes markedly in response to alterations of membrane potential. In the presence of an inwardly directed negative diffusion potential created by an inwardly directed thiocyanate (SCN-) gradient, the influx of lysine through this route is accelerated; with an inwardly directed positive potassium diffusion potential, lysine influx through this route is reduced. The influx of lysine through system y+L is much less sensitive to such alterations of potential. 4. Analysis of the kinetic constants characterizing system y+ shows that with a change of potential from zero to negative (approximately -60 mV) the maximum velocity (Vmax) is roughly doubled and the half-saturation constant (Km) halved leading to a 4-fold increase in permeability. For system y+L smaller changes are seen and Km does not change; the resulting increase in y+L permeability is 1.5-fold. 5. These findings are discussed with respect both to the mechanism of membrane transport and placental epithelial function.

Amino acid transport system y+L of human erythrocytes: specificity and cation dependence of the translocation step.

The transport specificity of system y+L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of L-[14C]-lysine. Some neutral amino acids, L-lysine and L-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external L-leucine and L-lysine increased the rate by 5.3 +/- 0.63 and 6.2 +/- 0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (L-tryptophan, L-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (L-alanine, L-serine). Half-saturation constants (+/- SEM) calculated from this effect (L-lysine, 10.32 +/- 0.49 microM and L-leucine, 11.50 +/- 0.50 microM) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na+, Li+ or K+ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na+ or Li+ were replaced by K+. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.

N-ethylmaleimide discriminates between two lysine transport systems in human erythrocytes.

1. The sulfhydryl reagent N-ethylmaleimide (NEM) was shown to inactivate the low affinity lysine transporter in human erythrocytes (system y+) without affecting the high affinity transporter (system y+L). 2. Pre-treatment of the cells with NEM reduced the rate of entry of L-[14C]lysine (1 microM) by approximately 50% (maximum effect). 3. NEM (0.2 mM) inhibited the NEM-sensitive component of the flux with mono-exponential kinetics. The inactivation rate constant (k, +/- S.E.M.) was 0.53 +/- 0.027 min-1 (25 degrees C). The substrate did not protect against inactivation. 4. Lysine self-inhibition experiments revealed two transport systems in untreated cells (half-saturation constants Km; +/- S.E.M.), 12.0 +/- 1.7 microM and 109 +/- 15.6 microM) and only one high affinity system in NEM-treated cells (Km 9.5 +/- 0.67 microM), indicating that NEM inactivates system y+. 5. The NEM-insensitive L-[14C]lysine influx (system y+L) was inhibited with high affinity by unlabelled neutral amino acids. The inhibition constant for L-leucine in sodium medium (Ki +/- S.E.M.) was 10.7 +/- 0.72 microM (37 degrees C). The system was also strongly inhibited by L-methionine, L-glutamine and with less affinity by L-phenylalanine and L-serine. N-methyl-L-leucine, L-proline and 2-amino-2-norbornane-carboxylic acid, a bicyclic analogue of leucine, did not exert a significant effect. 6. Lysine transport through system y+L occurred at the same rate in Na+, K+ or Li+ medium and the binding of lysine to the transporter was unaffected by Na+ replacement. 7. The interaction of system y+L with neutral amino acids was dependent on the cation present in the medium. The inhibition constant for leucine and glutamine increased approximately 90- and 60-fold respectively when Na+ was replaced by K+. Li+ was shown to be a very good substitute for Na+.

Identification of a new transport system (y+L) in human erythrocytes that recognizes lysine and leucine with high affinity.

1. The effect of neutral amino acids on the transport of L-lysine across the human erythrocyte membrane was studied. 2. All neutral amino acids tested (range 0.3-5 mM) inhibit the influx of L-[14C]lysine (1 microM). The inhibition pattern is biphasic, and tends to reach a maximum at approximately 50% of the original flux. The concentrations that give 25% inhibition are (mM): L-cysteine (2.7), L-alanine (1.3), L-serine (0.9), L-isoleucine (0.6), L-phenylalanine (0.35), L-methionine (< 0.3), L-leucine (< 0.3). L-lysine and L-arginine completely inhibit the rate at the highest concentration. 3. These results can be explained by assuming that L-lysine transport occurs through two independent transporters that differ in their affinity for neutral amino acids. A detailed kinetic analysis of the effect of L-leucine on L-lysine entry is consistent with this hypothesis. 4. Using a new experimental strategy, the substrate and inhibitor transport parameters for the two systems were determined. The half-saturation constants for lysine (+/- S.E.M.) are found to be: KmA, 0.014 +/- 0.002 mM and KmB, 0.112 +/- 0.017 mM. The maximum rates differ by a factor of 8.2 (VmaxB/VmaxA). The leucine inhibition constants are: KiA, 0.022 +/- 0.003 mM and KiB, 30.36 +/- 7.9 mM. If the sodium in the incubation medium is replaced by potassium, the apparent affinity for leucine (1/KiA) is reduced approximately 30-fold. 5. The maximum inhibition caused by leucine decreases as the lysine concentration is raised, showing that leucine acts upon the higher affinity system. 6. When added to the trans side, L-leucine, L-phenylalanine and L-isoleucine do not cause inhibition, but stimulate the flux by approximately 30%. This indicates that these analogues are also transported. 7. In conclusion, in the concentration range 1-100 microM, lysine crosses the red cell membrane through two distinct transport systems, one of which recognizes both neutral and cationic amino acids with high affinity.

A simple test for the sidedness of binding of transport inhibitors.

A new method is described for determining the sidedness of action of nonpolar inhibitors that rapidly diffuse through the lipid bilayer and could therefore interact with the carrier on both sides of the membrane. Sidedness is deduced from the effect of the inhibitor on the flux ratio for the substrate (the ratio of the rates of exchange and net transport). The advantages of the method are that the experimental measurements are made after the inhibitor has equilibrated rather than in the brief period when it is present on only one side of the membrane, and that any reversible inhibitor can be tested, whether the inhibition mechanism is competitive, noncompetitive, uncompetitive, or mixed.

Inhibition of choline transport in erythrocytes by n-alkanols.

The choline transport system of erythrocytes is reversibly inhibited by ethanol, n-butanol, n-hexanol, n-octanol, and n-decanol, but not by n-dodecanol. Each methylene group in the alkyl chain contributes 560 cal/mol to the free energy of binding at the inhibitory site. Inhibition results from the cooperative binding of two molecules of an alcohol, judging by the Hill coefficient n of 1.7-1.9. The mechanism of inhibition is noncompetitive, and the partition of the carrier between inward-facing and outward-facing forms is unaffected by the alcohols; it follows that the four main carrier forms, the inner and outer free carrier, and the inner and outer carrier-substrate complex, are equally susceptible to inhibition. Hexanol and decanol accelerate the reaction of N-ethylmaleimide with a thiol group in the inner carrier channel, but ethanol and butanol, at concentrations that inhibit transport by 70%, do not. The disproportionate effects on substrate transport and the N-ethylmaleimide reaction are most simply explained as the direct result of binding of alcohol molecules in different regions of the carrier, rather than as the indirect result of a disturbance in the structure of the lipid bilayer induced by the alcohols.

The determination of kinetic parameters for carrier-mediated transport of non-labelled substrate analogues: a general method applied to the study of divalent anion transport in placental membrane vesicles.

A method is described that allows the determination of kinetic parameters for carrier-mediated transport of unlabelled compounds. The ability of these compounds to relieve the inhibition of labelled substrate efflux produced by addition of an external competitive inhibitor is studied. The method is of general applicability and does not depend upon any intrinsic asymmetry in the ratio of the rates of translocation of the loaded and unloaded carrier. In this paper it is used in a study of the human placental sulphate transporter. Experiments on brush-border membrane vesicles show the method to predict quantitatively kinetic parameters for unlabelled sulphate entry. Further analysis shows this carrier to have a broad specificity for other oxyanions (selenate, tung-state, molybdate and chromate) with the following selectivity sequence: for rate of translocation, SO4, SeO4 greater than WO4 greater than MoO4 much greater than CrO4; for binding affinity, CrO4 greater than MoO4, WO4 greater than SO4, SeO4.