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Int J Biochem Cell Biol
2008 Jan 01;404:721-30. doi: 10.1016/j.biocel.2007.10.010.
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Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1.
Pieri M
,
Hall D
,
Price R
,
Bailey P
,
Meredith D
.
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The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen, as well as the oral absorption of a number of drugs, including beta-lactam antibiotics and angiotensin-converting enzyme inhibitors. Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains. Mutation of rabbit PepT1 arginine282 (R282, transmembrane domain 7) to a positive (R282K) or physiologically titratable residue (R282H), resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes. Neutral (R282A, R282Q) or negatively charged (R282D, R282E) substitutions gave a transporter that was not stimulated by external acidification (reducing pH(out) from 7.4 to 5.5) but transported at the same rate as the wild-type maximal rate (pH(out) 5.5); however, only the R282E mutation was unable to concentrate substrate above the extracellular level. All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type, except R282E-PepT1 which was faster. A conserved negatively charged residue, aspartate341 (D341) in transmembrane domain 8 was implicated in forming a charge pair with R282, as R282E/D341R- and R282D/D341R-PepT1 had wild-type transporter characteristics. Despite their differences in ability to accumulate substrate, both R282E- and R282D-PepT1 showed an increased charge:peptide stoichiometry over the wild-type 1:1 ratio for the neutral dipeptide Gly-l-Gln, measured using two-electrode voltage clamp. This extra charge movement was linked to substrate transport, as 4-aminobenzoic acid, which binds but is not translocated, did not induce membrane potential depolarisation in R282E-expressing oocytes. A model is proposed for the substrate binding/translocation process in PepT1.
Fig. 1. pH dependence of initial rate [3H]-d-Phe-l-Gln uptake by Xenopus oocytes expressing R282 mutants of rabbit PepT1, expressed as -fold stimulation by extracellular acidification (pHout 7.4 to 5.5), the dotted line showing where there is no stimulation (n ≥ 3 oocyte preparations with at least 5 oocytes per data point for each, except for R282Q-PepT1 where n = 2; *p < 0.05 for stimulation by external acidification, one-way ANOVA).
Fig. 2. Inhibition of [3H]-d-Phe-l-Gln uptake by increasing concentrations of Gly-l-Gln (0–1 mM) into Xenopus oocytes expressing either the various R282 mutants or wild-type PepT1 at pHout 5.5. Ki values were wild-type PepT1 0.39 ± 0.22, R282E 0.35 ± 0.17, R282K 0.48 ± 0.26, R282D 0.34 ± 0.27, R282A 0.30 ± 0.17, R282Q 0.46 ± 0.22 and R282H 0.30 ± 0.15 mM. n = 5 oocytes per data point.
Fig. 3. Accumulation of [3H]-d-Phe-l-Gln above the extracellular concentration by Xenopus oocytes expressing R282 mutants of rabbit PepT1. Fig. 3a and b shows representative time-course experiments at pHout 5.5 and 7.4, respectively. Fig. 3c and d shows the accumulation ratios at pHout 5.5 and 7.4 after 8 h incubation, respectively. The dotted line represents the equilibrium value if the volume of an oocyte is assumed to be 1 μl (*p < 0.05, one-way ANOVA). For R282E-PepT1 at pHout 7.4, the same level of accumulation was reached after 24 h incubation (2.3 ± 0.4-fold, p > 0.05, one-way ANOVA, n = 5 oocyte preparations with 5 oocytes per data point for each) as for the wild-type and the other mutants (see Section 3 for further details). NI = non-injected control oocytes.
Fig. 4. Efflux of intracellular [3H]-d-Phe-l-Gln in the presence of 5 mM Gly-l-Gln expressed as a fraction of that seen in the absence of trans-stimulant for non-injected control oocytes (NI), wild-type and R282 mutant PepT1 expressing Xenopus oocytes. Efflux by R282E-PepT1 at 30 min was significantly faster than for the wild-type and other R282 mutant PepT1s (*p < 0.05, one-way ANOVA), whilst efflux from all of the PepT1 expressing oocytes was faster than that for non-injected. After 60 min, the effluxes from all the PepT1 expressing oocytes were not significantly different, whilst significantly faster than for non-injected controls (p < 0.05, one-way ANOVA). n = 5 oocytes per data point.
Fig. 5. Representative trace of membrane potential from an oocyte expressing wild-type PepT1 (Fig. 5a) or R282E-PepT1 (Fig. 5b) in the absence or presence of Gly-l-Gln, d-Phe-l-Gln and 4-AMBA (0.6 mM, 0.4 μM and 10 mM respectively, for 1 min each), at pHout 5.5. Non-injected control oocytes showed no response (data not shown), with similar responses seen for at least 3 oocytes.
Fig. 6. Charge:peptide stoichiometry of uptake of Gly-l-Gln in Xenopus oocytes expressing R282E-, R282D- or R282A-PepT1, normalised to that of the wild-type PepT1 being 1:1 as previously reported (Fei et al., 1994; Steel et al., 1997). The average error on the current measurements was 13 ± 3.8% (n = 3 oocytes per mutant) and on the uptakes 4.4 ± 0.7% (n = 5 oocytes per mutant); the error bars on the figure represent the extremes of the stoichiometry that would be calculated using these error values.
Fig. 7. pH dependence of initial rate [3H]-d-Phe-l-Gln uptake into oocytes expressing wild-type, R282E-, D341R-, R282E/D341R- and R282D/D341R-PepT1 expressed as -fold stimulation by extracellular acidification (pHout 7.4–5.5), the dotted line showing where there is no stimulation (*p < 0.05 for the -fold stimulation being greater than one, one-way ANOVA, n ≥ 3 oocyte preparations).
Fig. 8. Protection of PepT1 from diethylpyrocarbonate (DEPC) inhibition (1 mM, 15 min incubation) by Gly-l-Gln (GlyGln), N-Acetyl-Phe (AcPhe) and l-Ala-Tyramine, but not Tyr (all present at 5 mM during DEPC incubation period). [3H]-d-Phe-l-Gln uptake was measured over 1 h. *p < 0.05 when compared to wild-type PepT1 (one-way ANOVA, 5 oocytes per data point, n = 3 oocyte preparations). NI = non-injected control oocytes.
Fig. 9. Model for PepT1 substrate binding and translocation. Arrows represent salt bridges or hydrogen bonds between residues.
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