4.5 Article

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

Journal

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1778, Issue 4, Pages 1042-1050

Publisher

ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2007.12.026

Keywords

amino acid transport; PAT1; histidine residues; site directed mutagenesis; drug delivery; proline

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The proton-coupled amino acid transporter 1 (PAT 1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT 1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT(1) because the corresponding mutants H55A, H55N and H55E had no detectable L-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1. (c) 2008 Elsevier B.V. All rights reserved.

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