Pharmaceutical Research

, Volume 20, Issue 12, pp 1911–1916

Biophysical Evidence for His57 as a Proton-Binding Site in the Mammalian Intestinal Transporter hPepT1

  • Tomomi Uchiyama
  • Ashutosh A. Kulkarni
  • Daryl L. Davies
  • Vincent H. L. Lee
Article

DOI: 10.1023/B:PHAM.0000008036.05892.e9

Cite this article as:
Uchiyama, T., Kulkarni, A.A., Davies, D.L. et al. Pharm Res (2003) 20: 1911. doi:10.1023/B:PHAM.0000008036.05892.e9

Abstract

Purpose. 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 H57R- 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 (Imax= 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.

proton-binding sitedipeptide transporterhistidinesite-directed mutagenesistwo-electrode voltage clampXenopus oocyteHEK293 cells

Copyright information

© Plenum Publishing Corporation 2003

Authors and Affiliations

  • Tomomi Uchiyama
    • 1
  • Ashutosh A. Kulkarni
    • 1
  • Daryl L. Davies
    • 2
  • Vincent H. L. Lee
    • 1
  1. 1.Department of Pharmaceutical SciencesUniversity of Southern CaliforniaLos Angeles
  2. 2.Department of Molecular Pharmacology and ToxicologyUniversity of Southern CaliforniaLos Angeles