Proton coupling and charge movement of the protondf.penuent oligopeptide transporter PEPT1

The mammalian proton-coupled oligopeptide transporter, PepTl (Nature 368:563-566, 1994) mediates peptide transport across intestinal epithelia. \ 'sing two-electrode voltage-clamp analysis of PepTl cRNA injected Xenopus oocyles we determined the apparent affinities and transport rates of neutral, acidic and basic dipeptides at different extracellular H⁺ concentrations (pH_o). Maximal transport and highest substrate affinity occurred at pH0 5.5, 5.2 and 6.2 for GlyLeu. Glyülu and GlyLys, respectively. Uptake of these peptides induced intracellular acidification as monitored by pH sensitive microelectrodes, indicating that the uptake of both neutral and charged dipeptides is coupled to the cotransport of H⁺. Analysis of the rate of intracellular acidification induced by neutral, acidic and basic peptides, in combination with measurements of the uptake rates of radiolabelled peptides and the substrate evoked charge fluxes, suggests proton to substrate coupling ratios of 1:1, 2:1 and 1:1. respectively. In oocytes injected with PepTl cRNA, but not water, capacitive and transient currents in response to step changes in membrane potential in the absence of substrate revealed a strong dependence of these currents on intra- and extracellular pH. V_1/2 , the voltage at which charge movements were half completed, varied linearly with the H⁺ equilibrium potential. One interpretation is that the observed transient currents are due to movement of a proton within the PepT1 transporter pore.