Expression cloning of a cDNA from rabbit small intestine related to proton-coupled transport of peptides, beta-lactam antibiotics and ACE-inhibitors

Abstract

Injection of poly(A)+ RNA from rabbit small intestine into Xenopus laevis oocytes resulted in expression of pH dependent transport of the aminocephalosporin cefadroxil. A cDNA library constructed from a 2.2 to 5 kb fraction was screened for expression of cefadroxil transport after injection of the corresponding cRNA synthetized in vitro from different pools of clones. The single clone identified stimulated uptake of cefadroxil into oocytes about 50-fold at pH 6.5. Kinetic analysis of expressed transport activity revealed a saturable transport system shared by amino beta-lactam antibiotics, dipeptides and selected angiotensin converting enzyme inhibitors. Evidence for rheogenic cefadroxil/H(+)-cotransport was obtained by a) The demonstration that cefadroxil influx increased the inward current in oocytes clamped at a holding potential of -60 mV in sodium-free medium and b) A decrease of intracellular pH in oocytes caused by cefadroxil uptake. Current-voltage relationships in the presence of glycylsarcosine or cefadroxil showed that transport activity is dependent on the membrane potential. Sequencing of the cDNA revealed its identity with the recently cloned peptide transporter from rabbit small intestine designated PepT1.