Isolation and function of the amino acid transporter PAT1 (slc36a1) from rabbit and discrimination between transport via PAT1 and system IMINO in renal brush-border membrane vesicles

Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H(+)/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5' and 3' untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na(+)-independent uptake of proline, glycine, l-alanine and alpha-(methylamino)isobutyric acid. Proline uptake was maximal at pH 5.0 (K(m) 2.2+/-0.7 mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na+ but in the presence of an inwardly directed H+ gradient. In the presence of Na+ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Structure,tissue expression pattern,and function of the amino acid transporter rat PAT2

The second member of the PAT (proton-coupled amino acid transporter) family of H(+)-coupled, pH-dependent, Na(+)-independent amino acid transporters was isolated from a rat lung cDNA library. The cDNA for rat PAT2 is 2396bp in length, including 70bp of 5'UTR and a poly(A) tail. The transporter gene, consisting of 10 exons, is located on rat chromosome 10q22. The cDNA codes for a protein of 481 amino acids with 72% identity (over 449 amino acids) with rat PAT1. Tissue expression studies demonstrate that mRNA abundance is generally low with highest levels being detected in lung and spleen, with lower levels in the brain, heart, kidney, and skeletal muscle. Functional expression in either mammalian cells or Xenopus laevis oocytes demonstrates that rat PAT2 mediates pH-dependent, Na(+)-independent uptake of glycine, proline, and alpha(methyl)aminoisobutyric acid (MeAIB). In conclusion PAT2 has a limited tissue distribution, higher affinity (Michaelis-Menten constant for glycine uptake between 0.49 and 0.69mM), and distinct substrate specificity compared to PAT1.     

Transport of l-proline by the proton-coupled amino acid transporter PAT2 in differentiated 3T3-L1 cells

Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 μM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate l-[³H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H⁺ gradient-stimulated uptake of l-[³H]proline. Uptake was rapid, independent of a Na⁺ gradient but stimulated by an inwardly directed H⁺ gradient with maximal uptake occurring at pH 6.0. l-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K_t) of 130 ± 10 μM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min^?1 mg of protein^?1. Glycine, l-alanine, and l-tryptophan strongly inhibited l-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H⁺-amino acid cotransport system PAT2.     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

The proton/amino acid cotransporter PAT2 is expressed in neurons with a different subcellular localization than its paralog PAT1

The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H⁺ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H⁺-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H⁺ in the course of cellular amino acid uptake by PAT1.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Transport of β-alanine in renal brush border membrane vesicles

The findings that the equilibrium uptake of β-alanine decreased with increasing medium osmolarity and preincubation with β-alanine increased uptake of the amino acid indicate that the uptake of β-alanine by rabbit renal brush border membranes represents transport into membrane vesicles. A Na⁺ electrochemical gradient (extravesicular > intravesicular) stimulated the initial rate of β-alanine uptake about three times and effected a transient accumulation of the amino acid twice the equilibrium value. Stimulation of the uptake was specific for Na⁺. Gramicidin abolished the overshoot, presumably by dissipating the gradient by accelerating the electrogenic entrance of Na⁺ into the vesicle via a pathway not coupled to uptake of β-alanine. In K⁺-loaded vesicle, valinomycin enhanced the Na⁺ gradient-dependent uptake of β-alanine. These findings indicate that the Na⁺ gradient-dependent transport of β-alanine is an electrogenic process and suggest that the membrane potential is a determinant of β-analine transport. Uptake of β-aniline, at a given concentration, reflected the sum of contributions from Na⁺ gradient-dependent and -independent transport systems. The dependent system saturated at 100 μM. The independent system did not saturate. At physiological concentrations the rate of the Na⁺ gradient-dependent uptake was four times that in the absence of the gradient. The Na⁺ gradient-dependent rate of β-alanine uptake was strongly inhibited by taurine, suggesting that β-amino acids have a common transport system, α-Amino acids, i.e. l-arginine, l-glutamate, l-proline, and glycine, representing previously reported specific α-amino acid transport systems in the brush border membrane, did not inhibit the uptake of β-alanine. These findings indicate that the brush border membrane has a distinct transport system for β-amino acids.     

Transport of L-proline,L-proline-containing peptides and related drugs at mammalian epithelial cell membranes

Summary. Membrane transport of L-proline has received considerable attention in basic and pharmaceutical research recently. Of the most recently cloned members of the solute carrier family, two are “proline transporters”. The amino acid transporter PAT1, expressed in intestine, kidney, brain and other organs, mediates the uptake of proline and derivatives in a pH gradient-dependent manner. The Na⁺-dependent proline transporter SIT1, cloned in 2005, exhibits the properties of the long-sought classical IMINO system. Proline-containing peptides are of interest for several reasons. Many biologically important peptide sequences contain highly conserved proline residues. Xaa-Pro peptides are very often resistant to enzymatic hydrolysis and display, in contrast to Pro-Xaa peptides, a high affinity to the H⁺/peptide cotransporter PEPT1 which is expressed in intestinal, renal, lung and biliary duct epithelial cells. Furthermore, several orally available drugs are recognized by PEPT1 as Xaa-Pro analogues due to their sterical resemblance to small peptides.     

Neutral amino acid transport by marine fish intestine: role of the side chain

This work was devoted to the study of the structure-affinity relationships in neutral amino acid transport by intestinal brush border of marine fish (Dicentrarchus labrax). The effects of the length of the side chain on kinetics of glycine, alanine, methionine and amino isobutyric acid were investigated. In the presence of K⁺ two components were characterized: one is saturable by increased substrate concentrations, whereas the other can be described by simple diffusion mechanism. Simple diffusion, a passive, non-saturable, Na⁺-independent route, contributes largely to the transport of methionine and to a much lesser extend to alanine, glycine or alphaaminoisobutyric acid uptakes. If a branched chain is present, as in the case of amino isobutyric acid, diffusion is low. A Na⁺-independent, saturable system has been fully characterized for methionine, but not for branched amino acids such as amino isobutyric acid. In the presence of Na⁺ saturable components were shown. Two distinct Na⁺-dependent pathways have been characterized for glycine uptake, with low and high affinities. For alanine and methionine only one Na⁺-dependent high affinity system exists with the same half-saturation concentration and the same maximum uptake at saturable concentrations. Glycine high affinity system has the same half-saturation concentration as methionine or alanine uptake, whereas maximum uptake is lower. The substitution of the hydrogen by a methyl group results in a severe decrease of uptake (aminoisobutyric acid). Mutual inhibition experiments indicate that the same carriers could be responsible for methionine and alanine uptakes and probably glycine Na⁺-dependent uptake. The influence of Na⁺ concentrations (100^-1 mol·l^-1) on amino acid uptake was examined. Glycine, alanine, methionine and amino isobutyric acid transport can be described by a hyperbolic function, with a saturation uptake which is highly increased for methionine. However, the half-saturation concentration does not seem to be strongly affected by the amino acid structure. The effect of Na⁺ concentration (25 and 100 mmol·l^-1) on the kinetics of methionine uptake have been also examined. The maximum uptake of the saturable system clearly shows a typical relationship with concentration.Abbreviations [AA] amino acid concentration - AIB aminoisobutyric acid - [I] Inhibitor amino acid concentration - J _i uptake in the presence of inhibitor - J _o uptake without inhibitor - K _d passive diffusion constant - K _i inhibitor constant - K _t concentration of test amino acid for half-maximal flux - MES 2[N-morpholino]ethanesulphonic acid - V _max maximum uptake at saturable amino acid concentrations - V _tot total amino acid uptake     

Uptake of proline by renal brushborder vesicles: A mathematical analysis

A mathematical model for amino acid uptake by membrane vesicles is described which includes two components, a Na⁺ dependent and a Na⁺ independent system. Uptake in the model is a function of both initial external Na⁺ and amino acid concentrations. Sodium dependence of amino acid transport in the model is manifested by changing affinity constants for amino acid uptake under different Na⁺ concentrations. The differing affinities for influx and efflux caused by increasing internal Na⁺ concentrations with time during transport incubations result in an “overshoot” for amino acid accumulation. For inwardly directed Na⁺ gradients, the model predicts the dependence of the occurrence of the overshoot on initial external substrate concentration and the dependence of the height of the overshoot on initial external Na⁺ concentration. This model has been used to describe experimental data on proline uptake by rat renal brushborder membrane vesicles.     

Uptake of selenate and selenite by isolated intestinal brush border membrane vesicles from pig,sheep, and rat

Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane Na⁺ gradient (Na _out ⁺ >Na _in ⁺ ). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na⁺ gradient. Unlike selenate uptake, selenite uptake was not Na⁺ dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of⁷⁵Se to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na⁺-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na⁺-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane Na⁺ gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of⁷⁵Se from⁷⁵Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups.     

DEVELOPMENTAL TRANSITIONS IN UPTAKE OF AMINO ACIDS BY SYNAPTOSOMAL FRACTIONS ISOLATED FROM RAT CEREBRAL CORTEX

Developmental changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Well-defined changes over an age continuum could be observed in both the rates of amino acid accumulation and the effects of Na⁺ on the accumulation. The uptakes of five amino acids (threonine, serine and valine in Na⁺-free medium, aspartic acid and proline in Na⁺-containing medium) increased progressively with the age of the animal, whereas the uptakes of leucine and arginine (in Na⁺-free medium) decreased steadily. The uptake of serine or threonine by synaptosomal fractions prepared from newborn rats was markedly dependent on the presence of Na⁺in the incubation media. Na⁺exerted progressively less effect on the accumulation process with continuing postnatal development and to some extent inhibited uptake by fractions obtained from rats older than about 15 days. Na⁺significantly enhanced the accumulation of glycine in fractions from newborn and adult rats, but had only a slight effect in fractions prepared from 12 to 17-day old rats. A detailed study of the accumulation of glycine indicated that the synaptosomal transport of this amino acid proceeded by two independent systems, one of which was totally dependent on external Na⁺and the and adult animals than in fractions from 12 to 17-day-old rats, wheras the Na⁺-independent system was most active during this latter period of development. The decline in the Na⁺-independent accumulation of glycine from about the 15th day to adulthood was characterized by a decrease in the V_max. and an increase in the K_m.     

Mechanism of proline uptake by Chlorella vulgaris

An amino acid uptake system specific for glycine, alanine, serine and proline was induced by glucose in Chlorella vulgaris. The uptake system translocated the zwitterionic form of the amino acid. There was more than 100-fold accumulation which indicated a coupling to metabolic energy. The depolarization of the membrane potential during proline uptake and the sensitivity of its uptake rate to the membrane potential point to coupling with an ion flow. Inhibitors of plasmalemma-bound H⁺-ATPase inhibit proline uptake. These data are interpreted to mean that proline is taken up as a proton symport. In some Chlorella strains the proline-coupled H⁺ uptake could be measured with electrodes, but not in Chlorella vulgaris. There is evidence that the transport of amino acids rapidly stimulates the proton-translocating ATPase of Chlorella vulgaris, so that the proline-coupled proton uptake is immediately neutralized.     

Sodium-coupled electrogenic transport of pyroglutamate (5-oxoproline) via SLC5A8, a monocarboxylate transporter

Pyroglutamate, also known as 5-oxoproline, is a structural analog of proline. This amino acid derivative is a byproduct of glutathione metabolism, and is reabsorbed efficiently in kidney by Na⁺-coupled transport mechanisms. Previous studies have focused on potential participation of amino acid transport systems in renal reabsorption of this compound. Here we show that it is not the amino acid transport systems but instead the Na⁺-coupled monocarboxylate transporter SLC5A8 that plays a predominant role in this reabsorptive process. Expression of cloned human and mouse SLC5A8 in mammalian cells induces Na⁺-dependent transport of pyroglutamate that is inhibitable by various SLC5A8 substrates. SLC5A8-mediated transport of pyroglutamate is saturable with a Michaelis constant of 0.36 ± 0.04 mM. Na⁺-activation of the transport process exhibits sigmoidal kinetics with a Hill coefficient of 1.8 ± 0.4, indicating involvement of more than one Na⁺ in the activation process. Expression of SLC5A8 in Xenopuslaevis oocytes induces Na⁺-dependent inward currents in the presence of pyroglutamate under voltage-clamp conditions. The concentration of pyroglutamate necessary for induction of half-maximal current is 0.19 ± 0.01 mM. The Na⁺-activation kinetics is sigmoidal with a Hill coefficient of 2.3 ± 0.2. Ibuprofen, a blocker of SLC5A8, suppressed pyroglutamate-induced currents in SLC5A8-expressing oocytes; the concentration of the blocker necessary for causing half-maximal inhibition is 14 ± 1 μM. The involvement of SLC5A8 can be demonstrated in rabbit renal brush border membrane vesicles by showing that the Na⁺-dependent uptake of pyroglutamate in these vesicles is inhibitable by known substrates of SLC5A8. The Na⁺ gradient-driven pyroglutamate uptake was stimulated by an inside-negative K⁺ diffusion potential induced by valinomycin, showing that the uptake process is electrogenic.     

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.     

Neutral amino acid transport in surface membrane vesicles isolated from mouse fibroblasts: Intrinsic and extrinsic models of regulation

Membrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated transport of several neutral amino acids into an osmotically-sensitive intravesicular space without detectable metabolic conversion of substrate. When a Na⁺ gradient, external Na⁺ > internal Na⁺, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6- to 9-fold above their external concentrations. Na⁺-stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na⁺-stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent K_m values for alanine and 2-aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system. Several observations indicated that both a Na⁺ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi-steady-state of accumulation were stimulated as a function of increasing concentrations of Na⁺ applied as a gradient (external > internal) across the membrane. This stimulation was independent of endogenous Na⁺, K⁺-ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na⁺. The apparent K_m for transport of alanine and 2-aminoisobutyric acid was decreased as a function of Na⁺ concentration. Similarly, in the presence of a standard initial Na⁺ gradient, quasi-steady-state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior-negative membrane potential imposed across the membrane by means of K⁺ diffusion potentials (internal > external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na⁺ and was blocked by the further addition of either nigericin or external K⁺. As a corollary, Na⁺-stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na⁺-coupled active transport of these amino acids but did not affect Na⁺-independent transport. Translocation of K⁺, H⁺, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na⁺ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na⁺, amino acid, and membrane component. Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40-transformed cells exhibited an increased Vmax of Na⁺-stimulated 2-aminoisobutyric acid transport, as well as an increased capacity for steady-state accumulation of amino acids in response t o a standard Na⁺ gradient, relative t o vesicles from nontransformed cells. Density-inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient-coupled transport systems are discussed.     

Substrate specificity of the amino acid transporter PAT1

Summary. The proton coupled amino acid transporter PAT1 expressed in intestine, brain, and other organs accepts L- and D-proline, glycine, and L-alanine but also pharmaceutically active amino acid derivatives such as 3-amino-1-propanesulfonic acid, L-azetidine-2-carboxylic acid, and cis-4-hydroxy-D-proline as substrates. We systematically analyzed the structural requirements for PAT1 substrates by testing 87 amino acids, proline homologs, indoles, and derivatives. Affinity data and effects on membrane potential were determined using Caco-2 cells. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate affinity. Hetero atoms in the proline template are well tolerated. Aromatic α-amino acids display low affinity. PAT1 interacts strongly with heterocyclic aromatic acids containing an indole scaffold. The structural requirements of PAT1 substrates elucidated in this study will be useful for the development of prodrugs.