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

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 (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.