The human intestinal di-/tripeptide transporter (hPEPT1) is a 12-transmembrane protein that facilitates transport of peptides from the intestine into the circulation. hPEPT1 is also an important target in oral delivery of drugs, but mechanistic and structural data for the protein are limited. In particular, there is little information on the function of the loops of the transporter. In this study, we show that mutation of several charged residues in the largest intracellular loop of hPEPT1 (loop 6–7, amino acids 224–278) significantly reduces hPEPT1 function. This loop has an asymmetric distribution of charged residues, with only positive charges in the N-terminal half and all five negative charges in the loop located in a small part of the C-terminal half. Point mutagenesis to alanine of three positive residues in the N-terminal half of loop 6–7 and four negative residues in the C-terminal half of the loop significantly reduced glycylsarcosine uptake. E267 was particularly sensitive to mutation, and kinetic analyses of E267A- and E267K-hPEPT1 gave V max values 10-fold lower than that for the wild-type protein. Secondary structure prediction suggested that loop 6–7 includes two amphipathic α-helices, with net positive and negative charges, respectively. We interpret the mutagenesis data in terms of interactions of the charged residues in loop 6–7 that may influence conformational changes of hPEPT1 during and after substrate transport.
The Journal of Membrane Biology – Springer Journals
Published: Nov 21, 2010
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