Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.
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 approximately 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-beta-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.
Boll
M
M
Institute of Nutritional Sciences, University of Giessen, Germany.
Herget
M
M
Wagener
M
M
Weber
W M
WM
Markovich
D
D
Biber
J
J
Clauss
W
W
Murer
H
H
Daniel
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002434:Cefadroxil / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D007421:Intestine, Small / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D009865:Oocytes; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D012333:RNA, Messenger; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; D002434:Cefadroxil