Cysteine scanning of transmembrane domain three of the human dipeptide transporter: implications for substrate transport.
The human intestinal dipeptide transporter (hPepT1) transports dipeptides and pharmacologically active drugs from the intestine to the blood. The role of transmembrane domain 3 (TMD3) of hPepT1 was studied using cysteine-scanning mutagenesis and methane thiosulfonate (MTS) cysteine modification. Each amino acid in TMD3 was individually mutated to a cysteine and Gly-Sar uptake by each mutated and modified transporter was determined relative to wild-type hPepT1. Uptake data for mutated transporters modified with the lipid-insoluble cysteine-modifying reagent MTSET suggested tilting of TMD3 relative to the substrate translocation pathway; the extracellular region of TMD3 showed little MTSET reactivity, indicative of solvent inaccessibility, whereas the intracellular part of TMD3 was relatively solvent accessible. Modification at 10 positions of TMD3 with MTSEA, a lipid-soluble cysteine-modifying reagent, gave unusual and statistically significant increases in Gly-Sar uptake relative to untreated mutants. We interpret these data in terms of the spatial properties of the hPepT1 substrate translocation channel and possible interactions of TMD3 with other transmembrane domains.
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Jennifer L S
JL
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089-9121, USA.
Kulkarni
Ashutosh A
AA
Davies
Daryl L
DL
Lee
Vincent H L
VH
Haworth
Ian S
IS
D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D002460:Cell Line; D003545:Cysteine / Q000235:genetics*; D004151:Dipeptides / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008698:Mesylates / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000074011:Peptide Transporter 1; D017354:Point Mutation; D013379:Substrate Specificity; D027981:Symporters / Q000235:genetics*; D014162:Transfection
D004151:Dipeptides; D008698:Mesylates; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; C093171:methanethiosulfonate ethylammonium; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C004194:glycylsarcosine; D005020:Ethyl Methanesulfonate; D003545:Cysteine