1. Kinetics of influx (mediated through peptide-proton cotransport) of two labelled dipeptides has been studied in apical membrane vesicles isolated from rat renal cortex. The substrates (neutral D-Phe-L-Ala and anionic D-Phe-L-Glu) have previously been shown to be transported through a single system but with different stoichiometry of proton coupling. 2. The initial rate of influx of both peptides was determined under a set of defined conditions allowing extravesicular pH, intravesicular pH, transmembrane pH and membrane potential (E(m)) to be varied systemically and independently. From this data the kinetic constants K(m) and V(max) were derived for each condition. Very substantial effects of pH, pH gradient and membrane potential were found; there were consistent quantitative differences when the substrates were compared. 3. Efflux of the two peptides from preloaded vesicles was also determined. At pH 5.5 (intra and extrasresicular), but not at pH 7.4, the rate constants for efflux of the two peptides mere similar and addition to the extravesicular medium of unlabelled D-Phe-L-Glu (but not D-Phe-L-Ala) trans-stimulated efflux of both peptides to a similar extent; the extent of this trans-stimulation was insensitive to alterations in membrane potential. 4. A model based on a combination of classical carrier theory (the carrier being negatively charged) and of two sequential protonation steps (both to external sites predicted to be in the membrane electrical field) is described. Qualitatively this adequately accounts for all the observations made and allows for the dependence of the stoichiometry of proton-peptide coupling on the net charge carried by the substrate. Quantitatively a 50-fold greater rate of reorientation of the free carrier when unprotonated is predicted to be responsible for the coupling of proton and peptide transport. 5. Our results and the model are discussed with respect to the recently elucidated primary structure of mammalian peptide transporters.
|Number of pages||14|
|Journal||Journal of Physiology|
|Publication status||Published - 1 Aug 1996|