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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 2, 2004 as doi:10.1096/fj.03-1387fje. |
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Molecular Nutrition Unit, Institute of Nutritional Sciences, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany
1Correspondence: Center of Life and Food Sciences, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany. E-mail: daniel{at}wzw.tum.de
SPECIFIC AIMS
We recently analyzed the structural requirements in substrates for binding and transport by the proton-dependent electrogenic amino acid transporters of the newly identified PAT family. Since a free amino group in a substrate was found not to be essential for substrate recognition and transport by the PAT1 and PAT2 proteins, we hypothesized that the transporters may be able to recognize and transport the corresponding physiologically important oxo acids acetate, propionate, and butyrate. Combining flux studies with radiolabeled oxo acids, the 2-electrode voltage clamp technique, and intracellular pH recordings in Xenopus oocytes, we characterized the transport of the oxo acids by PAT1 and PAT2.
PRINCIPAL FINDINGS
1. Amino acids but not short-chain fatty acids show electrogenic transport by the PAT proteins
The monocarboxylates acetate, propionate, and butyrate did not induce inward currents in oocytes expressing PAT2 or PAT2 but inhibited dose-dependently inward currents elicited by glycine, alanine, or proline. Elongation of the carbon chain in a short-chain fatty acid from acetate to butyrate resulted in increased inhibitory potency on glycine-mediated transport currents of PAT1 and PAT2, whereas octanoic acid exhibited only weak inhibitory potency. PAT1-mediated glycine currents recorded under voltage clamp conditions were reduced by propionate with an apparent Ki of 9.6 ± 0.9 mM. Similar results were obtained in flux studies with the tracer amino acid [3H]L-proline. Acetate, propionate, and butyrate decreased significantly PAT1- and PAT2-mediated uptake of labeled proline; kinetic analysis revealed for all three short-chain fatty acids typical first-order competition kinetics. Proline uptake by PAT1 was inhibited by acetate, propionate, and butyrate with apparent Ki values of 7.9 ± 0.5 mM, 12.0 ± 1.6 mM, and 5.6 ± 0.7, respectively. For PAT2, butyrate revealed a Ki of 7.6 ± 1.3 mM.
2. Despite the absence of transport currents, uptake of radiolabeled short-chain fatty acids is increased in oocytes expressing PAT1 or PAT2
To investigate whether the PAT-proteins mediate an electroneutral SCFA transport into oocytes, uptake of [3H]acetate and [14C]propionate (100 µM) was determined in the absence and presence of the PAT-substrate D-proline. Control oocytes possess a high rate of acetate uptake most likely by non-ionic diffusion. Expression of PAT1 increased acetate influx to 130% of control rates and PAT2 led to a 2.3-fold increase in acetate influx. In the presence of 20 mM D-proline, the PAT-specific increase in acetate influx was completely blocked and did not differ from that in control oocytes (144.8±5.1 pmol/oocyte/5 min), suggesting that D-proline and acetate share the same transport pathway. Confirmation was obtained using propionate as a tracer. In PAT1-expressing oocytes, propionate uptake was increased to 127% that in control oocytes and uptake in PAT2 oocytes by 150%.
3. Intracellular acidification during transport of short- chain fatty acids reveals evidence for electroneutral proton-dependent SCFA uptake
To prove the assumption that PAT proteins transport ionized species of the SCFA in symport with one proton in an electroneutral manner, we determined the corresponding changes in intracellular pH during SCFA uptake into oocytes under voltage clamp conditions. In response to acetate, propionate and butyrate transport of SCFA by PAT1 caused a sustained and faster decline in pHin than in water-injected control oocytes. Figure 1
A shows the changes in intracellular pH compared with current responses. SCFA did not cause an inward current but reduced significantly pHin, whereas glycine induced a strong inward current in PAT1-expressing oocytes with a concomitant reduction in intracellular pH. Figure 2B
compiles the initial acidification rates induced by SCFA or glycine in oocytes expressing PAT1 vs. those to control oocytes. Slopes of the PAT1-mediated decline in pHin in response to SCFA perfusion were 2.5- (propionate) and 2.8-fold (butyrate) higher than those obtained in control oocytes. The glycine-induced decrease in intracellular pH in oocytes expressing PAT1 occurred with essentially the same slope as that induced by butyrate, suggesting similar maximal transport rates for amino and oxo acids. That PAT1 mediates the strong initial acidification in PAT1-expressing oocytes in the presence of SCFA is demonstrated by a linear correlation between glycine-induced charge movement and acidification rates (Fig. 1C
).
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CONCLUSIONS
Transport of amino acids through cell membranes is realized by a multitude of membrane transporters. Using the proton-motive force for nutrient uptake is characteristic for transport processes in prokaryotes and simple eukaryotes. With the PAT-proteins, the first mammalian rheogenic proton-dependent amino acid transporters have been found. We present data that may assign a new function to mammalian PAT transporters, which are able to transport electrogenically small neutral amino acids such as glycine, alanine, and proline and the homologous oxo acids in an electroneutral mode and thereby are bifunctional.
We demonstrate that short chain fatty acids up to a chain-length of 6 carbons are competing substrates for uptake by PAT1 and PAT2. In contrast, octanoic acid exhibits only weak inhibitory effects, and lactate and pyruvate do not interfere with the substrate binding site of the PAT carriers showing that only short-chain and unsubstituted monocarboxylic acids are recognized. Using radiolabeled acetate and propionate, we show that expression of PAT1 and PAT2 in oocytes increases SCFA uptake and that this influx is inhibited by amino acids. However, electrophysiology failed to demonstrate any associated transport currents when SCFA are substrates but recordings of intracellular pH changes established that SCFA uptake is associated with proton influx under voltage clamp conditions and strongly suggests that SCFA-influx occurs by proton symport most likely by transport of the SCFA-anion with a proton in a 1:1 flux coupling stoichiometry, as shown for proline (Fig. 2
). Initial rates of intracellular acidification by PAT1 caused by butyrate or glycine uptake were similar whereas in propionate, acidification was 2-fold higher than with glycine. Acetate induced 2-fold higher acidification rates than glycine, suggesting that the maximal transport rates by which PAT1 mediates propionate and acetate uptake are different from those of glycine. Acetate, propionate, and butyrate displayed concentration-dependent inhibition of amino acid influx; apparent Ki values (5.6–12.0 mM) for PAT1 are in the same range as the apparent Km (1–10 mM) found for its amino acid substrates. PAT2 displays affinities in the range of 100 to 700 µM for glycine, alanine, proline but displays lower affinities for SCFA. This confirms the observation that PAT2 is more restrictive with regard to the structural requirements in substrates for high-affinity recognition than PAT1.
Transmembrane transport of SCFAs is assumed to occur by non-ionic diffusion, SCFA/anion exchange, and SCFA/proton symport mechanism. Permeation of the non-ionized species of a SCFA occurs rapidly by its apolar character, followed by fast intracellular dissociation. The driving force is the transmembrane concentration gradient of the SCFA and the transmembrane pH-gradient. The quantitative importance of this process compared with carrier-mediated SCFA uptake mechanisms is controversial.
In colonic tissues, various studies have assessed the transport mode for SCFA produced in huge quantities by bacterial fermentation, and it is thought that SCFA transport occurs via an anion/SCFA exchange mechanism. SCFA uptake into isolated proximal colonic membrane vesicles was increased in the presence of outwardly directed bicarbonate, butyrate, or propionate gradients and Ussing chamber experiments revealed that SCFAs are transported into the tissue via a bicarbonate/SCFA exchange mechanism. A proton/SCFA symport mechanism was proposed. Studies of the intestinal epithelial cell line Caco-2 and in isolated epithelial cells from rabbit proximal colon provided evidence for such a proton/SCFA cotransport system.
Until recently, only two transporter proteins mediating uptake of SCFAs have been characterized at the molecular level: monocarboxylate cotransporter MCT1 (SLC16A1) and the newly identified sodium-coupled monocarboxylate transporter SMCT (SLC5A8). MCT1 has been demonstrated to catalyze the proton-coupled transport of monocarboxylates such as lactate, pyruvate, acetate, propionate, and butyrate and ketone bodies. MCT1 is expressed in almost all tissues inter alia in cecum, colon, and kidney cortex. Whether MCT1 is localized in the apical or basolateral membrane of epithelial cells is not clear, but it appears to represent the basolateral form of the MCT series. The recently cloned SMCT transporter in contrast mediates Na+-coupled SCFA uptake and is expressed at high levels in colonic tissue. It transports lactate, pyruvate, acetate, butyrate, and propionate by an electrogenic Na+:SCFA cotransport mechanism.
We add two new protein entities to the list of membrane carriers that can transport selected SCFA. The PAT1-mRNA is abundantly expressed, with almost the same intensity along the gastrointestinal tract between stomach and descending colon, except in cecum. The PAT1 protein was recently shown to localize to the apical membrane of cells of the human intestinal epithelial cell line Caco-2. Acetate, propionate, and butyrate are extensively produced by microbial fermentation of nondigestible carbohydrates in colon and are present in the lumen in concentrations of 70–120 mM. Whether PAT1 in intestinal tissues contributes to overall SCFA uptake into the organism is not known. SCFAs are metabolized by colonic epithelial cells as a prime energy source, but portal blood of different species has concentrations of SCFAs from 0.4 and 5.3 mM, and SCFAs delivered to circulation serve as metabolic fuel for the liver and other peripheral tissues. It appears to be too soon to hypothesize that the PAT proteins play a role in transport of short-chain fatty acids or that SCFA modulate PAT-mediated amino acid transport in these tissues and cell types.
We demonstrate that mammalian PAT proteins 1 and 2 are novel bifunctional proton-coupled symporters that operate in electrogenic mode with amino acid substrates such as glycine, alanine, praline, or GABA or in an electroneutral mode when substrates lack an amino group. PAT-mediated proton/SCFA symport suggests a new function of these transporters in mammalian physiology.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1387fje;
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pH in PAT1-expressing (black line) and control oocytes (gray line) clamped at –40 mV. Fast pH changes at the beginning and end of glycine application are virtual and reflect potential differences between the tips of voltage recording and pH microelectrode. Lower traces represent the holding current recorded for a PAT1-expressing oocyte. B) Initial acidification rates in PAT1-expressing (black symbols) and control oocytes (gray symbols) during perfusion with propionate, butyrate, or glycine. Slope of total acidification was determined after linear regression of the first 20 s of linear acidification recorded in PAT1 and control oocytes. Data represent means ± SE of n = 10 experiments. C) Acidification rates of propionate (squares), butyrate (circles), and glycine (triangles) obtained in individual oocytes as a function of charge movement induced by glycine in the same oocyte at the end of each experiment.