Stefan Bröer

Amino acid transport across mammalian intestinal and renal epithelia.

The transport of amino acids in kidney and intestine is critical for the supply of amino acids to all tissues and the homeostasis of plasma amino acid levels. This is illustrated by a number of inherited disorders affecting amino acid transport in epithelial cells, such as cystinuria, lysinuric protein intolerance, Hartnup disorder, iminoglycinuria, dicarboxylic aminoaciduria, and some other less well-described disturbances of amino acid transport. The identification of most epithelial amino acid transporters over the past 15 years allows the definition of these disorders at the molecular level and provides a clear picture of the functional cooperation between transporters in the apical and basolateral membranes of mammalian epithelial cells. Transport of amino acids across the apical membrane not only makes use of sodium-dependent symporters, but also uses the proton-motive force and the gradient of other amino acids to efficiently absorb amino acids from the lumen. In the basolateral membrane, antiporters cooperate with facilitators to release amino acids without depleting cells of valuable nutrients. With very few exceptions, individual amino acids are transported by more than one transporter, providing backup capacity for absorption in the case of mutational inactivation of a transport system.

Comparison of lactate transport in astroglial cells and monocarboxylate transporter 1 (MCT 1) expressing Xenopus laevis oocytes: Expression of two different monocarboxylate transporters in astroglial cells and neurons

The transport of lactate is an essential part of the concept of metabolic coupling between neurons and glia. Lactate transport in primary cultures of astroglial cells was shown to be mediated by a single saturable transport system with aK m value for lactate of 7.7 mm and aV max value of 250 nmol/(min × mg of protein). Transport was inhibited by a variety of monocarboxylates and by compounds known to inhibit monocarboxylate transport in other cell types, such as α-cyano-4-hydroxycinnamate andp-chloromercurbenzenesulfonate. Using reverse transcriptase-polymerase chain reaction and Northern blotting, the presence of mRNA coding for the monocarboxylate transporter 1 (MCT1) was demonstrated in primary cultures of astroglial cells. In contrast, neuron-rich primary cultures were found to contain the mRNA coding for the monocarboxylate transporter 2 (MCT2). MCT1 was cloned and expressed in Xenopus laevis oocytes. Comparison of lactate transport in MCT1 expressing oocytes with lactate transport in glial cells revealed that MCT1 can account for all characteristics of lactate transport in glial cells. These data provide further molecular support for the existence of a lactate shuttle between astrocytes and neurons.

Transfer of glutamine between astrocytes and neurons

The export of glutamine from astrocytes, and the uptake of glutamine by neurons, are integral steps in the glutamate‐glutamine cycle, a major pathway for the replenishment of neuronal glutamate. We review here the functional and molecular identification of the transporters that mediate this transfer. The emerging picture of glutamine transfer in adult brain is of a dominant pathway mediated by system N transport (SN1) in astrocytes and system A transport (SAT/ATA) in neurons. The participating glutamine transporters are functionally and structurally related, sharing the following properties: (a) unlike many neutral amino acid transporters which have proven to be obligate exchangers, these glutamine transporters mediate net substrate transfer energized by coupling to ionic gradients; (b) they are sensitive to small pH changes in the physiological range; (c) they are susceptible to adaptive and humoral regulation; (d) they are related structurally to the AAAP (amino acid and auxin permeases) family of transporters. A key difference between SN1 and the SAT/ATA transporters is the ready reversibility of glutamine fluxes via SN1 under physiological conditions, which allows SN1 both to sustain a glutamine concentration gradient in astrocytes and to mediate the net outward flux of glutamine. It is likely that the ASCT2 transporter, an obligate exchanger of neutral amino acids, displaces the SN1 transporter as the main carrier of glutamine export in proliferating astrocytes.

Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes

Observations on lactate transport in brain cells and cardiac myocytes indicate the presence of a high-affinity monocarboxylate transporter. The rat monocarboxylate transporter isoform MCT2 was analysed by expression in Xenopus laevis oocytes and the results were compared with the known characteristics of lactate transport in heart and brain. Monocarboxylate transport via MCT2 was driven by the H(+) gradient over the plasma membrane. Uptake of lactate strongly increased with decreasing pH, showing half-maximal stimulation at pH 7.2. A wide variety of monocarboxylates and ketone bodies, including lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, 2-oxoisovalerate and 2-oxoisohexanoate, were substrates of MCT2. All substrates had a high affinity for MCT2. For lactate a K(m) value of 0.74+/-0.07 mM was determined at pH 7.0. For the other substrates, K(i) values between 100 microM and 1 mM were measured for inhibition of lactate transport, which is about one-tenth of the corresponding values for the ubiquitously expressed monocarboxylate transporter isoform MCT1. Monocarboxylate transport via MCT2 could be inhibited by alpha-cyano-4-hydroxycinnamate, anion-channel inhibitors and flavonoids. It is suggested that cells which express MCT2 preferentially use lactate and ketone bodies as energy sources.

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B0. We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G → A and 0.001 for 718C → T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.

SLC6 neurotransmitter transporter family

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Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation

The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mm) glutamine to displace 13C label supplied as [3‐13C]pyruvate, [2‐13C]acetate, l‐[3‐13C]lactate, or d‐[1‐13C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2‐(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1‐13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.

Effects of the Serine/Threonine Kinase SGK1 on the Epithelial Na+ Channel (ENaC) and CFTR: Implications for Cystic Fibrosis

Cystic fibrosis (CF) is characterized by impaired Cl- secretion and increased Na+ reabsorption in several tissues including respiratory epithelium. Many CFTR mutations have been identified over the past years. However, only a poor correlation between the genotype and lung phenotype was found suggesting additional factors influencing the phenotype and course of the disease. The serine/threonine kinase SGK1 has recently been shown to stimulate the activity of the epithelial Na+ channel ENaC. A variety of stimuli such as aldosterone, cell shrinkage, insulin or TGF-β1 stimulate transcription and activate the SGK1 kinase. Here we further examined the effects of SGK1 on ENaC and CFTR which have mutual interactions and we analyzed sgk1 mRNA abundance in lung tissue from CF patients. Coexpression of CFTR and h-SGK1 in Xenopus oocytes increased ENaC currents as previously described. In addition CFTR mediated currents were also stimulated. h-SGK1 accelerated the expression of the amiloride sensitive Na+- current in Xenopus oocytes paralleled by increased ENaC-protein abundance in the oocyte membrane, an effect which was reversed by a h-SGK1K127R mutation lacking the ATP-binding site. The cation selectivity or Na+ affinity were not affected. However, coexpression of h-SGK1 with ENaC altered the sensitivity of the Na+-channel to the inhibitors amiloride and triamterene. The inhibitory effect of CFTR expression on ENaC current was not affected by coexpression of h-SGK1 in Xenopus oocytes. Lung tissue from CF patients strongly expressed the serine/threonine kinase h-sgk1 which was not the case for non-CF lung tissue. Loss of CFTR function itself in a CF lung epithelial cell line did not increase SGK1 expression. In summary, enhanced expression of h-SGK1 in epithelial cells of CF-lung tissue may be a novel pathophysiological factor contributing to increased Na+ channel activity and thus to increased Na+ transport in CF. .

Comparison of fluorotyrosines and methionine uptake in F98 rat gliomas

The transport mechanisms of O-(2-[(18)F]fluoroethyl)-L-tyrosine (FET) and 2-[(18)F]fluoro-L-tyrosine (FTyr) were compared to those of [(3)H]-Methyl-L-methionine (MET) in F98 rat glioma cells in vitro and by tumor imaging by ex vivo dual tracer autoradiography in F98 rat gliomas. Both, FET and FTyr exhibited similar transport characteristics in F98 glioma cells compared to MET, i.e. mainly a sodium dependent transport similar to system B(0,+) and sodium independent transport via system L. Radioactivity of FET in the acid precipitable fraction was <1% after 120 min incubation time while FTyr and MET exhibited a 15-18% incorporation into proteins. Comparison of FET and FTyr with MET uptake in F98 rat gliomas demonstrated a significant correlation of tumor to brain ratios and a similar intratumoral tracer distribution pattern.

Adaptation of plasma membrane amino acid transport mechanisms to physiological demands

Abstract. The molecular identification of almost all physiologically characterized amino acid transporters in recent years has facilitated the functional analysis of this important class of transport proteins. The picture that emerges from these studies is that antiport is the prevalent mode of amino acid transport rather than a combination of uniporters and cotransporters. Mainly neurotransmitters and osmolytes are transported by complex cotransport mechanisms that allow a high intracellular accumulation. Antiport mechanisms almost invariably include the nonessential amino acids alanine and glutamine, which are used as exchange substrates. The intracellular level of both amino acids is well regulated by Na+/amino acid cotransporters. Transport mechanisms are not conserved within families and may change with mutation of even a single amino acid residue in the transport protein. Thus transport mechanisms are easily adapted to physiological demands during evolution.