Cystinuria-specific rBAT(R365W) mutation reveals two translocation pathways in the amino acid transporter rBAT-b0,+AT

Apical reabsorption of dibasic amino acids and cystine in kidney is mediated by the heteromeric amino acid antiporter rBAT/b 0,+ AT (system b 0,+ ). Mutations in rBAT cause cystinuria type A, whereas mutations in b 0,+ AT cause cystinuria type B. b 0,+ AT is the catalytic subunit, whereas it is believed that rBAT helps the routing of the rBAT/b 0,+ AT heterodimeric complex to the plasma membrane. In the present study, we have functionally characterized the cystinuria-specific R365W (Arg 365 →Trp) mutation of human rBAT, which in addition to a trafficking defect, alters functional properties of the b 0,+ transporter. In oocytes, where human rBAT interacts with the endogenous b 0,+ AT subunit to form an active transporter, the rBAT(R365W) mutation caused a defect of arginine efflux without altering arginine influx or apparent affinities for intracellular or extracellular arginine. Transport of lysine or leucine remained unaffected. In HeLa cells, functional expression of rBAT(R365W)/b 0,+ AT was observed only at the permissive temperature of 33 °C. Under these conditions, the mutated transporter showed 50% reduction of arginine influx and a similar decreased accumulation of dibasic amino acids. Efflux of arginine through the rBAT(R365W)/b 0,+ AT holotransporter was completely abolished. This supports a two-translocation-pathway model for antiporter b 0,+ , in which the efflux pathway in the rBAT(R365W)/b 0,+ AT holotransporter is defective for arginine translocation or dissociation. This is the first direct evidence that mutations in rBAT may modify transport properties of system b 0,+ .