Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

Abstract

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.

δ-Aminolevulinic acid transport in murine mammary adenocarcinoma cells is mediated by beta transporters

“…Novotny et al (2000) also reported that PEPT2 is one of the two transporters responsible for ALA incorporation at the choroid plexus. Results here presented indicate that in our murine mammary adenocarcinoma cell cultures ALA is not incorporated by any of these transporters since their substrates, the dipeptide gly-gly, the tripeptide gly-gly-ala and the angiotensinconverting enzyme inhibitor captopril (Boll et al, 1996) do not significantly block ALA uptake. Washed cells were preincubated for 15 min with 2 mM ouabaine or 0.1 mM dimethylamiloride or for 60 min with 100 mM 2-deoxy-D-glucose plus 10 mM Na 3 N. Then 0.1 mM 14 C-ALA or 0.1 mM 14 C-GABA was added and radioactivity was measured after 30 min.…”

Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1

“…Surprisingly, the corresponding histidine residues are also conserved in the putative membrane-spanning c~-helices of PEPT2 from the human [19], rabbit [28] and rat [22] kidney. Miyamoto et al [30] also reported that essential histidine residues might be present at the dipeptide-binding site of the rabbit renal dipeptide transporter.…”

“…Recent molecular approaches have revealed that two structurally different H+-coupled peptide transporters are localized in the intestine and kidney: PEPT1 is expressed primarily in the small intestine and to a lesser extent in the kidney [16,17,21], whereas the PEPT2 is expressed specifically in the kidney [19,22,28]. These transporters exhibit about 50% amino acid identity.…”

Immunolocalization and pharmacological relevance of oligopeptide transporter PepT1 in intestinal absorption of β‐lactam antibiotics

“…Enzyme assays rabbits, humans and rats [11][12][13][14][15][16], including a report from Alkaline phosphatase and Na+/K+-ATPase were assayed as deour laboratory [17]. The first report concerned rabbit intesscribed by Pekarthy et al [19] and Scharscbmidt et al [20], respectively.…”