PepT1 mediates the intestinal absorption and renal re-absorption of di- and tri-peptides and a great number of therapeutically active compounds (reviewed in Daniel et al., 2006). A 12 transmembrane domain (TMD) topology was proposed originally (Fei et al., 1994) and largely confirmed by epitope mapping, with the exception of TMDs 1&2 (Covitz et al., 1998). Recently, re-analysis of the sequence and homology modelling of PepT1 have suggested that TMD1 may have been mis-assigned (Meredith & Price, 2007). Rather than being amino acid residues 7-25, TMD1 is predicted by MEMSAT-3 to be formed from residues 24-42. Homology modelling suggests TMD1 will be tilted relative to the bilayer and therefore to be membrane spanning will need to be longer, and would be formed by residues 13-42. This re-assignment would put the conserved charged residues E23, E36 and R34 into TMD1; interestingly, E23 and E26 are conserved in one of the signature motifs for the dipeptide/tripeptide permease superfamily PTR2 (Daniel et al., 2006), while R34 is strongly conserved in higher organisms. Here we test whether these residues play a functional role in rabbit PepT1. The mutants E23R, E26R, R34E and R34Q were generated using a PCR based protocol on rabbit PepT1-FLAG epitope tagged template (wt-PepT1), and expressed in Xenopus oocytes. Both membrane expression and the transport properties were determined by luminometry and uptake of 0.4μM [³H]-D-Phe-L-Gln respectively using techniques previously described (Panitsas et al., 2006). Data are means ± SEM. As can be seen in Figure 1A, the uptake of D-Phe-L-Gln into oocytes expressing E23R-, E26R-, R34E- or R34Q-PepT1 at pH_out 5.5 was not significantly greater than that of non-injected oocytes, in contrast to those expressing wt-PepT1 (p>0.05, Student’s t-test, n=5 oocytes per data point, representative of up to 4 preparations). When the level of protein surface expression was quantified by luminometry (Figure 1B), it is clear that E23R- and E26R-PepT1 are not expressed at the membrane, whereas wt-PepT1, R34E- and R34Q-PepT1 were significantly expressed (p<0.001, n=12-18 oocyes per data point). These findings suggest that the residues mutated are crucial for functional rabbit PepT1 expression. E23 and E26 are implicated in proper protein folding/trafficking, as when mutated neither was expressed, whereas R34 is important for PepT1 function, as there was no dipeptide uptake despite membrane expression.