The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using the Xenopus laevis oocytes as an expression system and by combining the two-electron voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-alpha-amino acids substrates can consist maximally of only one CH2-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH2-units, as in gamma-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cystein only in the form of D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analyses of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations thet determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated.
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 ...
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