Biophysical Evidence for His57 as a Proton-Binding Site in the Mammalian Intestinal Transporter hPepT1
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- Uchiyama, T., Kulkarni, A.A., Davies, D.L. et al. Pharm Res (2003) 20: 1911. doi:10.1023/B:PHAM.0000008036.05892.e9
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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.