Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway

This study is the first systematic attempt to investigate the role of transmembrane segment 5 of hPepT1, the most conserved segment across different species, in forming a part of the aqueous substrate translocation pathway. We used cysteine-scanning mutagenesis in conjunction with the sulfhydryl-specific reagents, MTSEA and MTSET. Neither of these reagents reduced wild-type-hPepT1 transport activity in HEK293 cells and Xenopus oocytes. Twenty-one single cysteine mutations in hPepT1 were created by replacing each residue within TMS5 with a cysteine. HEK293 cells were then transfected with each mutated protein and the steady-state protein level, [H-3]Gly-Sar uptake activity, and sensitivity to the MTS reagents were measured. S164C-, L168C-, G173C-, and I179C-hPepT1 were not expressed on the plasma membrane. Y167C-, N171C-, and S174C-hPepT1 showed less than or equal to25% Gly-Sar uptake when compared with WT-hPepT1. P182C-hPepT1 showed similar to40% specific activity whereas all the remaining transporters, although still sensitive to single cysteine mutations, exhibited more than 50% specific activity when compared to WT-hPepT1. The activity of F166C-, L176C-, S177C-, T178C-, I180C-, T181C-, and P182C-hPepT1 was partially inhibited, while the activity of F163C- and I170C-hPepT1 was completely inhibited by 2.5 mM MTSEA. F163C, I165C, F166C, A169C, I170C, S177C, T181C, and P182C were clearly accessible to 1 mM MTSET. Overall, these results suggest that TMS5 lines the putative aqueous channel and is slightly tilted from the vertical axis of the channel, with the exofacial half forming, a classical amphipathic alpha-helix and the cytoplasmic half being highly solvent accessible. (C) 2003 Elsevier Science (USA). All rights reserved.