Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1.
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.
Boll
Michael
M
Molecular Nutrition Unit, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany.
Foltz
Martin
M
Anderson
Catriona M H
CM
Oechsler
Carmen
C
Kottra
Gabor
G
Thwaites
David T
DT
Daniel
Hannelore
H
D026905:Amino Acid Transport Systems / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001622:Betaine / Q000378:metabolism; D018938:Caco-2 Cells; D003001:Cloning, Molecular; D005260:Female; D006801:Humans; D008564:Membrane Potentials; D009865:Oocytes; D011485:Protein Binding; D013379:Substrate Specificity; D027981:Symporters; D014981:Xenopus
D026905:Amino Acid Transport Systems; D000596:Amino Acids; C475905:SLC36A1 protein, human; D027981:Symporters; D001622:Betaine