The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di-and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0-to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes "70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-f3-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.
The cloned intestinal peptide transporter is capable of electrogenic H+-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5-8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H+/H3O+ cotransport may vary depending on pH.
We used oocytes of the South African clawed toad Xenopus laevis to express the three subunits of the epithelial Na+ channel from rat distal colon (rENaC). We combined conventional dual‐microelectrode voltage‐clamp with continuous capacitance (C m) measurements and noise analysis to evaluate the effects of cAMP and Ni2+ on rENaC. Control oocytes or rENaC‐expressing oocytes exhibited no spontaneous fluctuations in current. However, in rENaC‐expressing oocytes amiloride induced a marked plateau‐shaped rise of the power density spectra. Recordings using four different concentrations of amiloride revealed that the blocker–channel interactions were of the first order. A cocktail of the membrane permeant cAMP analogue chlorophenylthio‐cAMP and IBMX (cAMP cocktail) increased amiloride‐sensitive current (I ami) and conductance (G ami). Furthermore, C m was also increased following cAMP application, indicating an increase in plasma membrane surface area. Noise analysis showed that cAMP increased the number of active channels in the oocyte membrane while single‐channel current decreased. From these data we conclude that cAMP triggered exocytotic delivery of preformed rENaCs to the plasma membrane. Ni2+ (2.5 mM) inhibited about 60% of the rENaC current and conductance while C m remained unaffected. Noise analysis revealed that this inhibition could be attributed to a decrease in the apparent channel density, while single‐channel current did not change significantly. These observations argue for direct effects of Ni2+ on channel activity rather than induction of endocytotic removal of active channels from the plasma membrane.
Endogenous glucose uptake by the oocytes of Xenopus laevis consists of two distinct components: one that is independent of extracellular Na+, and the other one that represents Na+-glucose cotransport. The latter shows similar characteristics as 2 Na+-1 glucose cotransport of epithelial cells: The similarities include the dependencies on external concentrations of Na+, glucose, and phlorizin, and on pH. As in epithelial cells, the glucose uptake in oocytes can also be stimulated by lanthanides. Both the electrogenic cotransport and the inhibition by phlorizin are voltage-dependent; the data are compatible with the assumption that the membrane potential acts as a driving force for the reaction cycle of the transport process. In particular, hyperpolarization seems to stimulate transport by recruitment of substrate binding sites to the outer membrane surface. The results described pertain to oocytes arrested in the prophase of the first meiotic division; maturation of the oocytes leads to a downregulation of both the Na+-independent and the Na+-dependent transport systems. The effect on the Na+-dependent cotransport is the consequence of a change of driving force due to membrane depolarization associated with the maturation process.
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