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

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 site dipeptide transporter histidine site-directed mutagenesis two-electrode voltage clamp Xenopus oocyte HEK293 cells 

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REFERENCES

  1. 1.
    H. Daniel. Function and molecular structure of brush border membrane peptide/H+ symporters. J. Membr. Biol. 154:197-203 (1996).Google Scholar
  2. 2.
    I. Rubio-Aliaga and H. Daniel. Mammalian peptide transporters as targets for drug delivery. Trends Pharmacol. Sci. 23:434-440 (2002).Google Scholar
  3. 3.
    S. Theis, B. Hartrodt, G. Kottra, K. Neubert, and H. Daniel. Defining minimal structural features in substrates of the H(+)/peptide cotransporter PEPT2 using novel amino acid and dipeptide derivatives. Mol. Pharmacol. 61:214-221 (2002).Google Scholar
  4. 4.
    X. Z. Chen, A. Steel, and M. A. Hediger. Functional roles of histidine and tyrosine residues in the H(+)-peptide transporter PepT1. Biochem. Biophys. Res. Commun. 272:726-730 (2000).Google Scholar
  5. 5.
    A. K. Yeung, S. K. Basu, S. K. Wu, C. Chu, C. T. Okamoto, S. F. Hamm-Alvarez, H. von Grafenstein, W. C. Shen, K. J. Kim, M. B. Bolger, I. S. Haworth, D. K. Ann, and V. H. Lee. Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton-coupled dipeptide transporter (hPepT1). Biochem. Biophys. Res. Commun. 250:103-107 (1998).Google Scholar
  6. 6.
    M. B. Bolger, I. S. Haworth, A. K. Yeung, D. Ann, H. von Grafenstein, S. Hamm-Alvarez, C. T. Okamoto, K. J. Kim, S. K. Basu, S. Wu, and V. H. Lee. Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1. J. Pharm. Sci. 87:1286-1291 (1998).Google Scholar
  7. 7.
    F. G. Grillo and P. S. Aronson. Inactivation of the renal microvillus membrane Na+-H+ exchanger by histidine-specific reagents. J. Biol. Chem. 261:1120-1125 (1986).Google Scholar
  8. 8.
    R. Hori, H. Maegawa, M. Kato, T. Katsura, and K. Inui. Inhibitory effect of diethyl pyrocarbonate on the H+/organic cation antiport system in rat renal brush-border membranes. J. Biol. Chem. 264:12232-12237 (1989).Google Scholar
  9. 9.
    H. M. Said and R. Mohammadkhani. Folate transport in intestinal brush border membrane: involvement of essential histidine residue(s). Biochem. J. 290(Pt 1):237-240 (1993).Google Scholar
  10. 10.
    M. Brandsch, C. Brandsch, M. E. Ganapathy, C. S. Chew, V. Ganapathy, and F. H. Leibach. Influence of proton and essential histidyl residues on the transport kinetics of the H+/peptide cotransport systems in intestine (PEPT1) and kidney (PEPT2). Biochim. Biophys. Acta 1324:251-262 (1997).Google Scholar
  11. 11.
    Y. Miyamoto, V. Ganapathy, and F. H. Leibach. Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter. J. Biol. Chem. 261:16133-16140 (1986).Google Scholar
  12. 12.
    T. Terada, H. Saito, M. Mukai, and K. I. Inui. Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1. FEBS Lett. 394:196-200 (1996).Google Scholar
  13. 13.
    T. Terada, H. Saito, and K. Inui. Interaction of beta-lactam antibiotics with histidine residue of rat H+/peptide cotransporters, PEPT1 and PEPT2. J. Biol. Chem. 273:5582-5585 (1998).Google Scholar
  14. 14.
    Y. J. Fei, W. Liu, P. D. Prasad, R. Kekuda, T. G. Oblak, V. Ganapathy, and F. H. Leibach. Identification of the histidyl residue obligatory for the catalytic activity of the human H+/peptide cotransporters PEPT1 and PEPT2. Biochemistry 36:452-460 (1997).Google Scholar
  15. 15.
    D. Meredith, C. S. Temple, N. Guha, C. J. Sword, C. A. Boyd, I. D. Collier, K. M. Morgan, and P. D. Bailey. Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1. Eur. J. Biochem. 267:3723-3728 (2000).Google Scholar
  16. 16.
    D. L. Davies, T. K. Machu, Y. Guo, and R. L. Alkana. Ethanol sensitivity in ATP-gated P2X receptors is subunit dependent. Alcohol. Clin. Exp. Res. 26:773-778 (2002).Google Scholar
  17. 17.
    D. D. Loo, A. Hazama, S. Supplisson, E. Turk, and E. M. Wright. Relaxation kinetics of the Na+/glucose cotransporter. Proc. Natl. Acad. Sci. USA 90:5767-5771 (1993).Google Scholar
  18. 18.
    B. Mackenzie, D. D. Loo, Y. Fei, W. J. Liu, V. Ganapathy, F. H. Leibach, and E. M. Wright. Mechanisms of the human intestinal H+-coupled oligopeptide transporter hPEPT1. J. Biol. Chem. 271:5430-5437 (1996).Google Scholar

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

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