Abstract
To investigate the involvement of transmembrane segment 7 (TMS7) of hPepT1 in forming the putative central aqueous channel through which the substrate traverses, we individually mutated each of the 21 amino acids in TMS7 to a cysteine and analyzed the mutated transporters using the scanning cysteine accessibility method. Y287C- and M292C-hPepT1 did not express at the plasma membrane. Out of the remaining 19 transporters, three (F293C-, L296C-, and F297C-hPepT1) showed negligible glycyl-sarcosine (gly-sar) uptake activity and may play an important role in defining the overall hPepT1 structure. K278C-hPepT1 showed approximately 40% activity and the 15 other transporters exhibited more than 50% gly-sar uptake when compared with wild type (WT)-hPepT1. Gly-sar uptake for the 16 active transporters containing cysteine mutations was then measured in the presence of 2.5 mM 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 1 mM [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET). Gly-sar uptake was significantly inhibited for each of the 16 single cysteine mutants in the presence of 2.5 mM MTSEA. In contrast, significant inhibition of uptake was only observed for K278C-, M279C-, V280C-, T281C-, M284C-, L286C-, P291C-, and D298C-hPepT1 in the presence of 1 mM MTSET. MTSET modification of R282C-hPepT1 resulted in a significant increase in gly-sar uptake. To investigate this further, we mutated WT-hPepT1 to R282A-, R282E-, and R282K-hPepT1. R282E-hPepT1 showed a 43% reduction in uptake activity, whereas R282A- and R282K-hPepT1 had activities comparable with WT-hPepT1, suggesting a role for the Arg-282 positive charge in substrate translocation. Most of the amino acids that were MTSET-sensitive upon cysteine mutation, including R282C, are located toward the intracellular end of TMS7. Hence, our results suggest that TMS7 of hPepT1 is relatively solvent-accessible along most of its length but that the intracellular end of the transmembrane domain is particularly so. From a structure-function perspective, we speculate that the extracellular end of TMS7 may shift following substrate binding, providing the basis for channel opening and substrate translocation.
Highlights
It is mainly expressed in the small intestine [1], proximal tubules of the kidney [2], and in the lysosomes of liver cells [2], pancreatic cells [3], and renal cells [4]
K278C-hPepT1 showed ϳ40% activity and the 15 other transporters exhibited more than 50% gly-sar uptake when compared with wild type (WT)-hPepT1
Helical wheel analysis of the effects of cysteine-scanning and MTS modification in transmembrane segment 7 (TMS7) of hPepT1 show that the amino acid residues that are sensitive to MTS modification are positioned over the majority of the circumference of the wheel (Fig. 7). This is in sharp contrast with the results that we have reported for TMS5 of hPepT1 [29], in which the MTSET-sensitive residues are gathered together on a single face of a generally amphipathic ␣-helix [29]
Summary
NA AAG to TGC ATG to TGC GTT to TGT ACG to TGC AGG to TGT GTG to TGC ATG to TGC TTC to TGC CTG to TGC TAT to TGT ATT to TGC CCA to TGC CTC to TGC CCA to TGC ATG to TGC TTC to TGC TGG to TGC GCC to TGC TTG to TGC TTT to TGT GAC to TGC modifying reagents. We have shown previously that the 11 endogenous cysteines present in WT-hPepT1 either do not react with the MTS reagents or that their modification with the MTS reagents does not have a significant impact on hPepT1 activity [29] This makes the application of SCAM to hPepT1 appropriate. We have recently shown that TMS5 is an integral part of the hPepT1 substrate translocation pathway by using a systematic cysteine-scanning mutagenesis approach in conjunction with MTS reagents [29]. These results suggested that TMS5 is slightly tilted from the vertical axis of the channel, with the exofacial half-forming a classical amphipathic ␣-helix and the cytoplasmic half being highly solventaccessible [29]. Our cysteine-scanning data and further mutagenesis results for Arg-282 suggest that TMS7 is a relatively solvent-accessible transmembrane segment and is so at its cytoplasmic end
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
