Site-directed mutation of arginine 282 to glutamate uncouples the movement of peptides and protons by the rabbit proton-peptide cotransporter PepT1.
A conserved positive residue in the seventh transmembrane domain of the mammalian proton-coupled di- and tripeptide transporter PepT1 has been shown by site-directed mutagenesis to be a key residue for protein function. Substitution of arginine 282 with a glutamate residue (R282E-PepT1) gave a protein at the plasma membrane of Xenopus laevis oocytes that was able to transport the non-hydrolyzable dipeptide [3H]d-Phe-l-Gln, although unlike the wild type, the rate of transport by R282E-PepT1 was independent of the extracellular pH level, and the substrate could not be accumulated above equilibrium. The binding affinity of the mutant transport protein was unchanged from the wild type. Thus, R282E-Pept1 appears to have been changed from a proton-driven to a facilitated transporter for peptides. In addition, peptide transport by R282E-PepT1 still induced depolarization as measured by microelectrode recordings of membrane potential. A more detailed study by two-electrode voltage clamping revealed that R282E-PepT1 behaved as a peptide-gated non-selective cation channel with the ion selectivity series lithium > sodium > N-methyl-d-glucamine at pH 7.4. There was also a proton conductance (comparing pH 7.4 and 8.4), and at pH 5.5 the predominant conductance was for potassium ions. Therefore, it can be concluded that changing arginine 282 to a glutamate not only uncouples the cotransport of protons and peptides of the wild-type PepT1 but also creates a peptide-gated cation channel in the protein.
Meredith
David
D
Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, United Kingdom. david.meredith@anat.ox.ac.uk
D000818:Animals; D001120:Arginine; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D004594:Electrophysiology; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000074011:Peptide Transporter 1; D021381:Protein Transport; D011522:Protons; D011817:Rabbits; D027981:Symporters
D002352:Carrier Proteins; D000074011:Peptide Transporter 1; D011522:Protons; D027981:Symporters; D018698:Glutamic Acid; D001120:Arginine