title: Biophysical evidence for His57 as a proton-binding site in the mammalian intestinal transporter hPepT1.
abstract: OBJECTIVE
  The objective of this study was to provide direct evidence of the relative importance of the His57 residue present in transmembrane domain 2 (TMD 2) and the His121 residue in TMD 4 as proton-binding sites in human PepT1 (hPepT1) by using a novel mutagenesis approach.
  
  
  METHODS
  His57 and His121 in hPepT1 were each mutated to alanine, arginine, or lysine individually to obtain H57A-, H57R-, H57K-, H121A-, H121R-, and H121K-hPepT1. H7A-hPepT1 was used as a negative control. [3H]Glycylsarcosine (Gly-Sar) uptake was measured 72 h posttransfection using HEK293 cells individually transfected with these mutated proteins. Steady-state I/V curves (-150 mV to +50 mV, holding potential -70 mV) were obtained by measuring 5 mM Gly-Sar-induced currents in oocytes expressing H-57R- and H57K-hPepT1. Noninjected oocytes and wild-type hPepT1 (WT-hPepT1)-injected oocytes served as negative and positive controls, respectively.
  
  
  RESULTS
  At pH 6.0, H57K-, H57R-, H121K-, and H121R-hPepT1 led to a 97%, 90%, 45%, and 75% decrease in [3H]Gly-Sar uptake into HEK293 cells, respectively. At pH 7.4, uptake in cells transfected with H57K- and H57R-hPepT1 was not significantly different from that at pH 6.0, whereas cells expressing H121R- and H121K-hPepT1 showed 56% and 65% decrease, respectively, compared to that at pH 6.0. In oocytes expressing H57R-hPepT1, steady-state currents induced by 5 mM Gly-Sar increased with increasing pH (I(max) = 300 nA at pH 8.5), suggesting the binding of protons to H57R. No such trend was observed in oocytes injected with H57K, H121R, and H121K cRNA.
  
  
  CONCLUSIONS
  H57R-hPepT1 is able to bind protons at a relatively basic pH, resulting in facilitation of transport of Gly-Sar by hPepT1 at higher pH. Our novel approach provides direct evidence that His57 is a principal proton-binding site in hPepT1.
authors.affiliation: Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Ave., Los Angeles, CA 90089-9121, USA.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Uchiyama
authors.forename: Ashutosh A
authors.initials: AA
authors.lastname: Kulkarni
authors.forename: Daryl L
authors.initials: DL
authors.lastname: Davies
authors.forename: Vincent H L
authors.initials: VH
authors.lastname: Lee
mesh_terms: D000465:Algorithms; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D000074011:Peptide Transporter 1; D011522:Protons; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D027981:Symporters; D014162:Transfection; D014981:Xenopus
chemical_list: D002352:Carrier Proteins; D000074011:Peptide Transporter 1; D011522:Protons; C082186:SLC15A1 protein, human; D027981:Symporters; D006639:Histidine; D012313:RNA; D001120:Arginine; D008239:Lysine