Backgroung and aim: 

The broad range amino acid transporter B⁰AT1 (Slc6a19) interacts with two specific tissue accessory proteins, Collectrin in the kidney and its homolog the angiotensin converting enzyme 2 (ACE2) in small intestine. A strong down regulation of B⁰AT1 expression in the kidney and small intestine was observed in the collectrin and ace2 knock-out animals respectively. Additionally, a significant increase of the transporter function was observed when B⁰AT1 was co-expressed with Collectrin or ACE2 in oocytes of Xenopus laevis. To better understand the interaction between B⁰AT1 and Collectrin, as well as its impact on B⁰AT1 expression and stability we overexpressed B⁰AT1 alone or in the presence of the kidney specific accessory protein Collectrin in MDCK cells.

Results: 

Stable MDCK cells lines expressing B⁰AT1, Collectrin, or B⁰AT1 and Collectrin were first generated using a retroviral system. The integrity of monolayers formed by stably transfected cells was tested by measuring mannitol fluxes. The expression of the transporter or the accessory protein was assayed by immunofluorescence and the transport function was assayed by uptakes of radiolabeled amino acid. Interestingly, the MDCK cell line expressing B⁰AT1 and Collectrin did not form tight monolayers. B⁰AT1 as well as Collectrin localization appeared to be intracellular exhibiting a paranuclear staining pattern that evokes endoplasmatic and Golgi localization. However functional tests demonstrated a significant higher L-Ile uptake in B⁰AT1-Collectrin co-expressing cell lines than in wild type (wt) and in cell lines expressing separately B⁰AT1 or Collectrin. Taken together, these results imply that the expression of Collectrin plus B⁰AT1 interferes with normal cell differentiation and growth, suggesting the possibility that the induced uptake of amino acids and Na⁺ is toxic for these cells. To circumvent this problem, we utilized an inducible lentivirus system to express B⁰AT1 in the MDCK cells. We first produced a stable MDCK cell line expressing inducibly the repressor protein (KRAB). This cell line was used as acceptor for our gene of interest, B⁰AT1, expressed under the control of the KRAB transrepressor. We analyzed B⁰AT1 expression by q-PCR and immunofluorescence. Our preliminary results suggest that B⁰AT1 is expressed in the MDCK-KRAB cells only after induction. The electrical resistance and the phenotype of these cells were like the wild type.

Conclusion: 

The MDCK cells line inducibly expressing B⁰AT1 can now be used to study the interaction of this amino acid transport with its kidney specific accessory protein Collectrin without disturbing the homeostasis of the cells.