The epithelial electrogenic H+/peptide symporter PepT1 mediates the uptake of di-, tri-peptides and peptidomimetics in the intestine and kidney, reviewed by Meredith & Boyd (2000). A recent study (Meredith 2004) has shown that substitution of the conserved argine in position 282 (TM7) of PepT1 with a glutamate (R282E-PepT1) results in a transporter that is capable of transporting the dipeptide [³H]-D-Phe-L-Gln but unlike the wild type the uptake is independent of extracellular pH. Therefore Arg-282 is considered to be involved in the proton gating of the transporter, possibly by charge pairing to another negatively charged residue. Based on these findings we investigated which residue interacts with Arg-282. A conserved aspartic acid (D341, TM8) was considered a putative charge pair candidate due to its structural proximity, and its negative charge. A single D341R-PepT1 and a double R282E/D341R-PepT1 mutant were generated in rabbit PepT1 using site-directed mutagenesis (Quickchange, Stratagene). The two mutations were confirmed by DNA sequencing. cRNA was microinjected and the uptake of the hydrolysis-resistant dipeptide [³H]-D-Phe-L-Gln was measured as previously described (Meredith 2004). The controls employed were non-injected, wild type (WT)-PepT1 and R282E-PepT1 expressing oocytes. All data are normalized (pH 5.5 = 1) and expressed as mean ± S.E.M., n = 4-7 experiments of minimum 5 oocytes per experiment (Figure 1). The comparison between uptakes in different pH values was made using paired t-tests. Uptake of the dipeptide D-Phe-L-Gln into oocytes expressing the single D341R mutant was found to be affected by extracellular acidification in a manner similar to the WT-PepT1 (alkalization from pH 5.5 to pH 7.4 results in a 3-fold statistically significant reduction of D341R-PepT1 uptake, P < 0.005). The R282E-PepT1 uptake remained constant whilst the pH was increased, in agreement with the published data. Interestingly, the double mutant R282E/D341R-PepT1 appears to follow the pH dependency of the WT transporter (3-fold statistically significant reduction of uptake in pH 7.4 compared to uptake in pH 5.5, P < 0.005). Taken together, these results suggest that the pH independence of uptake by R282E-PepT1 is restored to a wild type like behaviour by the double mutation R282E/D341R-PepT1. They further suggest that introduction of the second mutation compensates for the R282E mutation by re-establishing a charge pair.