Cystinuria-specific rBAT(R365W) mutation reveals two translocation pathways in the amino acid transporter rBAT-b^0,+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 (Arg³⁶⁵ → 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 holotran sporter 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,+.