A translational approach to investigate the role of membrane transport proteins in the renal stone disease, cystinuria

Abstract

In the kidney, unbound amino acids are freely filtered into the lumen of the nephron. For reabsorption to occur, they must be transported across the phospholipid bilayers of the tubular epithelium by selective transport systems. Mutations in these transport systems can lead to disease though a conferred lack of amino acid re-absorption. One such disease is cystinuria, caused by mutations in SLC3A1 and SLC7A9, which encode the two protein subunits of System b0,+, rBAT and b0,+AT, respectively. In healthy individuals System b0,+ mediates Na+- independent reabsorption of dibasic amino acids, and the cysteine dimer, cystine, in exchange for neutral amino acids. In cystinuric patients, these amino acids are not sufficiently reabsorbed causing a dibasic aminoaciduria and the precipitation of cystine crystals, leading to the formation of renal calculi. A cohort of cystinuric patients was recruited to the study, and both genes were screened for causal variants. A range of techniques was employed to enable the detection of small point mutations and large genomic rearrangements. Four novel missense variants were detected in SLC3A1. These were M465K, N254T, L416P and Y579D. In silico homology modeling of rBAT against the crystal structure of B. cereus oligo-1,6-glucosidase (PDB code 1UOK), predicted the location of these mutations in the extracellular domain of the protein. When rBAT cRNA was injected into Xenopus oocytes, uptake of the prototypical System b0,+ substrate [3H]arginine was observed, following the association of human rBAT with an endogenous oocyte light chain. A series of techniques was optimised to allow the characterisation of FLAG-tagged rBAT function and expression in oocytes, 1-6 days postinjection of cRNA. Mutations in rBAT lead to a mis-folding of the protein and its early degradation in the ER, preventing successful trafficking of the System b0,+ heterodimer to the renal epithelial membrane. This aberrant trafficking leads to reduced rBAT expression and System b0,+ activity in oocytes. Functional characterisation of the novel mutant proteins led to a decrease in the Vmax of [3H]arginine transport. Over-expression of rBAT in oocytes apparently overcomes the defect and leads to a recovery of function over time. However, [3H]arginine uptake in M465Kexpressing oocytes was still lower than that observed with wild-type rBAT even at 6 days postinjection. These data were supported by immunofluorescent detection of rBAT and the mutant proteins at the plasma membrane of oocytes. Western blotting of total membrane proteins from oocytes expressing mutated rBAT showed decreased total protein, suggestive of an increased rate of degradation associated with the pathogenic variants. An increased understanding of the effect of these mutations on the biogenesis of rBAT will contribute to the identification of novel therapeutic targets in the treatment of cystinuria.