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

Abstract

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 degrees 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,+).

Expression of genes coding for selected amino acid transporters in small intestine, liver, and skeletal muscle of pigs fed excess branched-chain amino acids

“…Transporter b 0 , + AT is mainly expressed in epithelial cells and functions as an antiporter exchanging Leu for Lys so that the absorption of Lys is coupled with the efflux of Leu (Pineda et a., 2004). As reported previously Hatzoglou et al, 2004) AT expression in the ileum was not affected by excess Leu or LIV.…”

“…Transport of Lys across the apical membrane of enterocytes is primarily facilitated by the AA transporter b 0 , + AT and with lower activity by the CAT1 transport system. The transporter b 0 , + AT exchanges Lys for neutral AA, particularly Leu Majumder et al, 2009) AT that is coupled with the efflux of Leu (Pineda et al, 2004). We recently observed reduced expression of b 0 , + AT in growing pigs (García-Villalobos et al, 2012;Morales et al, 2013) fed excess Leu diets, and surplus Ile failed to correct the reduction (Cervantes-Ramírez et al, 2013).…”

Molecular Cloning of Mouse Amino Acid Transport System B0, a Neutral Amino Acid Transporter Related to Hartnup Disorder

“…12). Uptake of cationic amino acids in kidney and intestine is mediated by the heteromeric amino acid transporter rBAT/b 0 AT in exchange for neutral amino acids (41). As a result removal of neutral amino acids is thought to provide an additional driving force for the accumulation of cationic amino acids in the kidney (Fig.…”

Molecular Genetic Analysis of SLC3A1 and SLC7A9 Genes in Czech and Slovak Cystinuric Patients

“…Currently, more than 80 mutations in SLC3A1 and 50 mutations in SLC7A9 have been found in cystinuric patients (Calonge et al 1994;Gasparini et al 1995;Bisceglia et al 1996Bisceglia et al , 2001Gitomer et al 1998aGitomer et al , 1998bPras et al 1998;Saadi et al 1998;Boutros et al 1999;Feliubadaló et al 1999;Purroy et al 2000;Font et al 2001;Harnevik et al 2001;Botzenhart et al 2002;Leclerc et al 2002;Schmidt et al 2002Schmidt et al , 2003. Defective amino acid transport has been reported for eight of the missense mutations of SLC3A1, after expression in Xenopus laevis oocytes (Pineda et al 2004). However, complete characterization of the defective amino acid Here we present the results of mutation analysis in Czech and Slovak patients with cystinuria using direct sequencing of their SLC3A1 and SLC7A9 genes.…”