Substrate specificity and transport mode of the proton‐dependent amino acid transporter mPAT2

Abstract

The PAT2 transporter has been shown to act as an electrogenic proton/amino acid symporter. The PAT2 cDNA has been cloned from various human, mouse and rat tissues and belongs to a group of four genes (pat1 to pat4) with PAT3 and PAT4 still resembling orphan transporters. The first immunolocalization studies demonstrated that the PAT2 protein is found in the murine central nervous system in neuronal cells with a proposed role in the intra and/or intercellular amino acid transport. Here we provide a detailed analysis of the transport mode and substrate specificity of the murine PAT2 transporter after expression in Xenopus laevis oocytes, by electrophysiological techniques and flux studies. The structural requirements to the PAT2 substrates -when considering both low and high affinity type substrates -are similar to those reported for the PAT1 protein with the essential features of a free carboxy group and a small side chain. For high affinity binding, however, PAT2 requires the amino group to be located in an a-position, tolerates only one methyl function attached to the amino group and is highly selective for the L-enantiomers. Electrophysiological analysis revealed pronounced effects of membrane potential on proton binding affinity, but substrate affinities and maximal transport currents only modestly respond to changes in membrane voltage. Whereas substrate affinity is dependent on extracellular pH, proton binding affinity to PAT2 is substrate-independent, favouring a sequential binding of proton followed by substrate. Maximal transport currents are substrate-dependent which suggests that the translocation of the loaded carrier to the internal side is the rate-limiting step.Keywords: electrophysiology; functional characterization; proton symporter; substrate recognition; transport mode.The proton/amino acid transporter family PAT (SLC36) has been identified from human, mouse and rat origin during the last three years [1][2][3][4][5][6][7]. The PAT family is comprised of four members (PAT1-PAT4, SLC36A1-4); the PAT proteins consist of around 470-500 amino acids and are thought to represent integral membrane proteins with a > 60% similarity to each other [8]. The orthologous proteins from mouse, rat and human show an identity of more than 90% to each other. The expression pattern of the four different transporter mRNAs is quite different. The PAT1 mRNA shows widespread expression with high levels in brain, intestine and kidney whereas the PAT2 mRNA is highly abundant in lung, kidney and brain. PAT3 mRNA is found solely in testis, whereas PAT4 mRNA shows ubiquitous expression [8].So far, only PAT1 and PAT2 have been characterized functionally. They have an exceptional position among the mammalian amino acid transporters, as they act as electrogenic amino acid/proton symporters. Transport is dependent on the extracellular pH, but is independent of sodium, chloride, and potassium ions [1,2,4,7]. Moreover, PAT1 and PAT2 mediated amino acid influx leads to a pronounced intracellular acidification [2,9] by cotransport ...

Proton-dependent glutamine uptake by aphid bacteriocyte amino acid transporter ApGLNT1

“…Animals live in a microbial world, and many animals form long- 38 lasting beneficial associations with microbial partners that facilitate bio-39 diversity [1,2]. An estimated 10% of all insect species harbor obligate 40 bacterial endosymbionts in specialized cells and tissues [3].…”

“…AAAP family H + -coupled amino acid transporters from mammals 361 [30,38,39] and plants [40][41][42] that are related to ApGLNT1 are sensitive 362 to membrane potential. Here, we show that ApGLNT1-mediated gluta-363 mine currents are significantly altered by changes in membrane poten-364 tial ( Fig.…”

SLC36A4 (hPAT4) Is a High Affinity Amino Acid Transporter When Expressed in Xenopus laevis Oocytes

“…The main substrates for PAT2 are the small neutral amino acids glycine, alanine, and proline (17), which have a higher affinity than for PAT1 (11,14) (Table 1). PAT2 has a narrower substrate specificity than PAT1 (26); for example, although both ␤-amino and ␥-amino butyric acids can inhibit PAT1, they have no effect on PAT2 (11).…”

“…PAT2 mRNA shows a high abundance in organs such as the kidneys, lungs, and spinal cord and brain (14,26). The subcellular localization of PAT2 in vivo is still to be determined, but in HeLa cells, it does not localize to lysosomes but seems to be expressed in the plasma membrane (23).…”

“…PAT3 was first cloned from mice (12), and mRNA expression was found to be very high in the testis and negligible elsewhere (13). PAT4 was first identified based on sequence similarity to the remaining members of the SLC36 family and seems to have a more ubiquitous expression (14). Here we describe the results of experiments that characterize the function of hPAT4 (SLC36A4) when heterologously expressed in Xenopus laevis oocytes and show for the first time that it is an amino acid transporter with a very high affinity for proline and tryptophan.…”