Functional expression of the rabbit intestinal Na+/L-proline cotransporter(IMINO system) in Xenopus laevis oocytes

Proline absorption across small intestine takes place mainly through a Na+- dependent cotransporter localized at the brush border membrane of the enter ocyte named IMINO system. It transports L-proline and 4-OH-proline but not L-alanine, neither cationic nor anionic amino acids. The present work demon strates the functional expression of this transporter in Xenopus laevis ooc ytes by mRNA microinjection and radiotracer uptake techniques. Poly (A)(+)- RNA was isolated from rabbit jejunal mucosa and injected into oocytes. Five days after the injection, results showed 1.5 fold stimulation of 50 mu M H -3-proline uptake by the injected oocytes when compared to the non injected oocytes uptake. Poly (A)(+)-RNA was sized fractionated and fractions were injected again. Increase on Na+-dependent L-proline uptake was obtained wit h a mRNA fraction between 2,4 and 4,4 kb, which was used to construct a cDN A library. The library was sequentially divided and cRNAs injected into ooc ytes in order to screen for an increment on the signal. A subdivision conta ining around 2,000 colonies was found to augment L-proline uptake 25 fold o ver the non injected oocytes uptake. This cRNA pool was used to further cha racterize the transporter. Results showed that in the absence of Na+ there was no L-proline uptake, 2 mM 4-OH-L-proline completely inhibited 50 mu M p roline uptake and there was no 50 mu M alanine uptake. In summary, these re sults demonstrate the expression of the rabbit small intestine IMINO transp orter in Xenopus laevis oocytes and support the next steps in the isolation of the clone.