EXPRESSION CLONING OF A CDNA FROM RABBIT SMALL-INTESTINE RELATED TO PROTON-COUPLED TRANSPORT OF PEPTIDES, BETA-LACTAM ANTIBIOTICS AND ACE-INHIBITORS

Injection of poly(A)(+) RNA from rabbit small intestine into Xenopus l aevis oocytes resulted in expression of pH dependent transport of the aminocephalosporin cefadroxil. A cDNA library constructed from a 2.2 t o 5 kb fraction was screened for expression of cefadroxil transport af ter injection of the corresponding cRNA synthetized in vitro from diff erent pools of clones. The single clone identified stimulated uptake o f cefadroxil into oocytes about 50-fold at pH 6.5. Kinetic analysis of expressed transport activity revealed a saturable transport system sh ared by amino beta-lactam antibiotics, dipeptides and selected angiote nsin converting enzyme inhibitors. Evidence for rheogenic cefadroxil/H +-cotransport was obtained by a) The demonstration that cefadroxil inf lux increased the inward current in ooyctes clamped at a holding poten tial of-60 mV in sodium-free medium and b) A decrease of intracellular pH in oocytes caused by cefadroxil uptake. Current-voltage relationsh ips in the presence of glycylsarcosine or cefadroxil showed that trans port activity is dependent on the membrane potential. Sequencing of th e cDNA revealed its identity with the recently cloned peptide transpor ter from rabbit small intestine designated PepT1.