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Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1

Lookup NU author(s): Dr Catriona Anderson, Professor David Thwaites

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Abstract

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 Xenopus laevis oocytes as an expression system and by combining the two-electrode voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-α-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 γ-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cysteine only in the form of the 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 analysis 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 that determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and side chain size, whereas substitutions on the amino group are well tolerated.


Publication metadata

Author(s): Boll M, Foltz M, Anderson CMH, Oechsler C, Kottra G, Thwaites DT, Daniel H

Publication type: Article

Publication status: Published

Journal: Molecular Membrane Biology

Year: 2003

Volume: 20

Issue: 3

Pages: 261-269

Print publication date: 01/07/2003

ISSN (print): 0968-7688

ISSN (electronic): 1464-5203

Publisher: Informa Healthcare

URL: http://dx.doi.org/10.1080/0968768031000100759

DOI: 10.1080/0968768031000100759

PubMed id: 12893527


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