Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1.
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(out) 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 binding/translocation process in PepT1.
Pieri
Myrtani
M
Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford OX1 3QX, UK.
Hall
Dashiell
D
Price
Richard
R
Bailey
Patrick
P
Meredith
David
D
D000818:Animals; D001120:Arginine / Q000235:genetics*; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004594:Electrophysiology; D005260:Female; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000187:drug effects; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009865:Oocytes / Q000502:physiology; D000074011:Peptide Transporter 1; D010455:Peptides / Q000378:metabolism* / Q000493:pharmacokinetics; D011522:Protons; D011817:Rabbits; D027981:Symporters / Q000235:genetics* / Q000502:physiology; D014982:Xenopus laevis
D000074011:Peptide Transporter 1; D010455:Peptides; D011522:Protons; D027981:Symporters; D001120:Arginine; D004047:Diethyl Pyrocarbonate