1 Introduction Daniel & Kottra, 2004 Meredith & Boyd, 2000 Terada & Inui, 2004 Bailey et al., 2000; Bailey et al., 2006; Biegel et al., 2005 Covitz, Amidon, & Sadee, 1998 Bolger et al., 1998 Panitsas, Boyd, & Meredith, 2006 Meredith, 2004 Pieri, Boyd, & Meredith, 2004 2 Materials and methods 2.1 Site-directed mutagenesis of the PepT1 gene - 5' CGCAGATCAAGATGGTTACG xxx GTGCTGTTCCTGTACATCC 3' xxx CAA AAG GAT CAT GCG - 5' TCCTGGTCCCCATCATG xxx GCCGTGGTGTATCC 3' xxx CGC Reverse primers for the PepT1 mutant PCR reactions were the reverse compliment of the forward primers. The site-directed PepT1 mutants were generated using the Quikchange protocol (Stratagene), and the resulting constructs confirmed by DNA sequencing (Department of Biochemistry, University of Oxford, UK). 2.2 cRNA synthesis and oocyte injection in vitro X. laevis 4 3 2 −1 2.3 Transport experiments trans 3 d l Meredith, 2004 2 2 3 d l 3 d l 3 d l l K i Deves and Boyd (1989) Meredith, 2004 trans- l Terada, Saito, and Inui (1998) out l N Meredith et al., 2000 l 2.4 Electrophysiology d l l 2.5 Data analysis Fig. 6 Fei et al., 1994; Steel et al., 1997 2.6 Statistical analysis p 3 Results 3.1 d l Fig. 1 d l p Meredith, 2004 K i l 3 d l Fig. 2 3.2 Can the R282 mutant PepT1 transporters concentrate substrates? Meredith, 2004 Fig. 3 out Fig. 3 out Petersen & Berridge, 1996 Yao & Tsien, 1997 out Fig. 3 d l out out p 3.3 d l Fig. 4 3 d l p p Meredith, 2004 p p 3.4 Apparent transport stoichiometry (charge to substrate) using two-electrode voltage clamp Meredith, 2004 d l out out n Fig. 5 Fei et al., 1994; Steel et al., 1997 out Fig. 6 3.5 Is the extra current measured dependent on substrate transport? Darcel, Liou, Tome, & Raybould, 2005 Meredith et al., 1998 K i Meredith et al., 2000 l K i Fig. 5 3.6 Identification of an interacting residue for R282 Fig. 7 3.7 Diethylpyrocarbonate inhibition of PepT1 function Fig. 8 l N Meredith et al., 2000 K i l Fig. 8 4 Discussion K out out K Tishmack, Bashford, Harms, & Van Etten, 1997 K Meredith, 2004 Meredith, 2004 out Temple & Boyd, 1998 Meredith, 2004 Pieri et al., 2004 Meredith, 2004 Kulkarni et al. (2007) Kulkarni et al., 2007 Fig. 6 out out out Temple & Boyd, 1998 out in Temple, Bailey, Bronk, & Boyd, 1996 out in Panitsas et al., 2006 out out out out out out Temple et al., 1996 Bailey et al., 2000 Meredith & Boyd, 1995 Steel et al., 1997 Uchiyama, Kulkarni, Davies, & Lee, 2003 K a out out out out Fig. 9 + Meredith et al., 2000 + Terada et al. (1998) N Meredith et al., 2000 l + Fig. 8 + Yeung et al., 1998 K a Fig. 6 In conclusion, the arginine at position 282 in rabbit PepT1 plays an intriguing role in the function of the transporter, with mutations to different residues revealing that a positive residue is required for pH dependence, whilst only R282E-PepT1 cannot concentrate substrate above equilibrium; this is despite other mutations, most notably R282D-PepT1, having a similarly increased charge:peptide stoichiometry. As previously proposed, R282 (TMD7) forms a charge pair with D341 (TMD8), with R282E/D341R-PepT1 showing normal transport characteristics. Further biological testing or a crystal structure of PepT1 will be required to establish the validity of the model proposed.