Journal
INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
Volume 40, Issue 4, Pages 721-730Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.biocel.2007.10.010
Keywords
epithelia; membrane transport; SLC15a1; nutrient absorption; protein structure-function; site-directed mutagenesis
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Funding
- Wellcome Trust Funding Source: Medline
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The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen, as well as the oral absorption of a number of drugs, including beta-lactam. antibiotics and angiotensin-converting enzyme inhibitors. Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains. Mutation of rabbit PepT1 arginine282 (R282, transmembrane domain 7) to a positive (R282K) or physiologically titratable residue (R282H), resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes. Neutral (R282A, R282Q) or negatively charged (R282D, R282E) substitutions gave a transporter that was not stimulated by external acidification (reducing pH(out) from 7.4 to 5.5) but transported at the same rate as the wild-type maximal rate (pH,,ut 5.5); however, only the R282E mutation was unable to concentrate substrate above the extracellular level. All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type, except R282E-PepT1 which was faster. A conserved negatively charged residue, aspartate341 (D341) in transmembrane domain 8 was implicated in forming a charge pair with R282, as R282E/D341R- and R282D/D341R-PepT1 had wild-type transporter characteristics. Despite their differences in ability to accumulate substrate, both R282E- and R282D-PepT1 showed an increased charge:peptide stoichiometry over the wild-type 1: 1 ratio for the neutral dipeptide Gly-L-Gln, measured using two-electrode voltage clamp. This extra charge movement was linked to substrate transport, as 4-aminobenzoic acid, which binds but is not translocated, did not induce membrane potential depolarisation in R282E-expressing oocytes. A model is proposed for the substrate hinding/translocation process in PepT1. (C) 2007 Elsevier Ltd. All rights reserved.
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