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 enterocyte named IMINO system. It transports L-proline and 4-OH-proline but not L-alanine, neither cationic nor anionic amino acids. The present work demonstrates the functional expression of this transporter in Xenopus laevis oocytes 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 microM 3H-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 with a mRNA fraction between 2,4 and 4,4 kb, which was used to construct a cDNA library. The library was sequentially divided and cRNAs injected into oocytes in order to screen for an increment on the signal. A subdivision containing around 2,000 colonies was found to augment L-proline uptake 25 fold over the non injected oocytes uptake. This cRNA pool was used to further characterize 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 microM proline uptake and there was no 50 microM alanine uptake. In summary, these results demonstrate the expression of the rabbit small intestine IMINO transporter in Xenopus laevis oocytes and support the next steps in the isolation of the clone.
Urdaneta
E
E
Departamento de Fisiología y Nutrición, Facultad de Farmacia, Universidad de Navarra, Pamplona, Spain.
Barber
A
A
Wright
E M
EM
Lostao
M P
MP
D000409:Alanine / Q000378:metabolism; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D007413:Intestinal Mucosa / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism*; D011061:Poly A / Q000235:genetics; D011392:Proline / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity; D027981:Symporters; D014982:Xenopus laevis
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D027981:Symporters; D011061:Poly A; D011392:Proline; D012964:Sodium; D000409:Alanine