Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes

Abstract

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...

A novel nutrient sensing mechanism underlies substrate-induced regulation of monocarboxylate transporter-1

“…These results further implicate that butyrate elicits these effects extracellularly by activating sensors/receptors on the cell surface. Regulation of transporter function via specific sensing of luminal nutrient substrates has been described for the intestinal sugar transporter Glut2 (16,21), the peptide transporter PEPT1 (23), and, recently, also for the intestinal iron transporters DMT1 and ferroporin 1 (25). However, the underlying mechanisms and pathways are not fully understood.…”

“…The short-term regulation of the activity of a transporter protein may involve alterations of its membrane abundance or changes in some kinetic properties such as substrate affinity, interaction with other molecules, or ion dependence (23). Butyrate-induced activation of MCT1 function in polarized C2BBe1 cells was kinetically manifested by an increase in the V max , without altering the K m .…”

Temperature effects on the kinetic properties of the rabbit intestinal oligopeptide cotransporter PepT1

“…Briefly, to prepare the mRNA, the cDNA encoding the PepT1 transporters was cloned into the pSPORT-1 vector [18] and linearized with NotI for rbPepT1, and with HindIII for sbPepT1. cRNAs were then synthesized in vitro in the presence of Cap Analog and 200 units of T7 RNA polymerase.…”

Functional and structural determinants of reverse operation in the pH-dependent oligopeptide transporter PepT1

“…Briefly, 20 ng of the plasmid containing the FLAG-wild-type PepT1 cDNA [24] was amplified with 2.5 units of Pfu DNA polymerase in the presence of overlapping primers containing in their sequence the mutated codons:…”

“…In summary, to prepare the mRNA, the cDNA encoding PepT1-FLAG transporters, cloned into the pSPORT-1 vector [24], were linearized with NotI; subsequently cRNA was synthesized in vitro in the presence of Cap Analog and 200 units of T7 RNA polymerase. All enzymes were supplied by Promega Italia, Milan, Italy.…”