PAT1 is a recently identified member of the PAT family of proton/amino acid co-transporters with predominant expression in the plasma membrane of enterocytes and in lysosomal membranes of neurons. Previous studies in Xenopus oocytes expressing PAT1 established proton/substrate co-transport associated with positive inward currents for a variety of small neutral amino acids. Here we provide a detailed analysis of the transport mode of the murine PAT1 in oocytes using the two-electrode voltage-clamp technique to measure steady-state and pre-steady-state currents. The GPC (giant patch clamp) technique and efflux studies were employed to characterize the reversed transport mode. Kinetic parameters [K(m) (Michaelis constant) and I(max) (maximum current)] for transport of various substrates revealed a dependence on membrane potential: hyperpolarization increases the substrate affinity and maximal transport velocity. Proton affinity for interaction with PAT1 is almost 100 nM, corresponding to a pH of 7.0 and is independent of substrate. Kinetic analysis revealed that binding of proton most likely occurs before substrate binding and that the proton and substrate are translocated in a simultaneous step. No evidence for a substrate-uncoupled proton shunt was observed. As shown by efflux studies and current measurements by the GPC technique, PAT1 allows bidirectional amino acid transport. Surprisingly, PAT1 exhibits no pre-steady-state currents in the absence of substrate, even at low temperatures, and therefore PAT1 takes an exceptional position among the ion-coupled co-transporters.
Mertl M, Daniel H, Kottra G. Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes. Am J Physiol Cell Physiol 295: C1332-C1343, 2008. First published September 17, 2008 doi:10.1152/ajpcell.00241.2008.-The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of ϳ5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-L-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed when endocytosis was stimulated by the activation of PKC. Cytochalasin D inhibited all changes evoked by either dipeptide or PKC stimulation, whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substratedependent retrieval of rPEPT1. Coexpression experiments with human Na ϩ -glucose transporter 1 (hSGLT1) revealed that substrate exposure selectively affected PEPT1 but not the activity of hSGLT1. electrophysiology; dipeptide; presteady-state currents; surface expression; endocytosis THE CAPACITY OF A TRANSPORTER PROTEIN can be regulated either by variation of its membrane abundance or variation of its kinetic properties (e.g., turnover rate, substrate affinity, and ion dependency). Transporter density in the membrane usually involves exocytosis or endocytosis of the proteins delivered to or retrieved from the plasma membrane (26,29,33,53), whereas the kinetic properties can be regulated either by direct phosphorylation and dephosphorylation (8,28) or by the association of cytosolic regulatory proteins (46). A number of observations have suggested that substrates can, via nonkinetic mechanisms, also regulate the activity of their transporters, but the underlying signaling pathways that cause such substrate-dependent protein regulation are mostly unknown. Such transport regulation processes have even been observed in heterologous expression systems. Xenopus laevis oocytes exposed to a mixture of 20 amino acids at concentrations approximating those in Xenopus p...
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