Human cystinuria-related transporter: Localization and functional characterization

Background. Cystinuria has been proposed to be an inherited defect of apica l membrane transport systems for cystine and basic amino acids in renal pro ximal tubules, Although the mutations of the recently identified transporte r BAT1/b(0.+) AT have been related to nontype I cystinuria, the function an d localization of human BAT1 (hBAT1)/b(0.+) AT have not been well character ized. Methods. The cDNA encoding hBAT1 was isolated from human kidney. Fluorescen ce in situ hybridization was performed to map the hBAT1 gene on human chrom osomes. Tissue distribution and localization of expression were examined by North ern blot and immunohistochemical analyses. hBAT1 cDNA was transfecte d to COS-7 cells with rBAT cDNA, and the uptake and efflux of C-14-labeled amino acids were measured to determine the functional properties. The roles of protein kinase-dependent phosphorylation were investigated using inhibi tors or activators sf protein kinases. Results. The hBAT1 gene was mapped to 19q12-13.1 on the human chromosome, w hich is the locus of nontype I cystinuria. hBAT1 message was expressed pred ominantly in kidney. hBAT1 protein was localized in the apical membrane of proximal tubules in human kidney. When expressed in COS-7 cells with a type II membrane glycoprotein rBAT (related to b(0.+)-amino acid transporter), hBAT1 exhibited the transport activity with he properties of amino acid tra nsport system b(0.+), which transported cystine as well as basic and neutra l amino acids presumably via a substrate exchange mechanism. BAT-mediated t ransport was reduced by the protein kinase A activator and enhanced by the tyrosine kinase inhibitor. Conclusions. hBAT1 exhibited the properties expected for a transporter subs erving the high-affinity cystine transport system in renal proximal tubules . The hBAT1 gene was mapped to the locus of nontype I cystinuria, confirmin g the involvement of hBAT1 in cystinuria.