(A) Pedigrees consistent with autosomal recessive inheritance are shown. Filled symbols represent individuals with DA, arrows indicate each proband, and the diagonal line represents a deceased subject. Representative chromatograms from control or DA pedigree DNA, with allele genotype (WT, wildtype; Mut, mutant) indicated. Boxes indicate the location of each mutation. (B) RFLP analysis of DA pedigrees. For c.1333C > T RFLP, AgeI digestion releases 252- and 229-bp fragments (shown as control [C]). The c.1333C > T mutation inactivates the AgeI site, leaving a 481-bp product (top panel). For c.1184–1186delTCA RFLP, BsaBI does not cut the 461-bp PCR product (shown as control). The c.1184-1186delTCA mutation results in a 458-bp amplicon, which is cut by BsaBI into 245- and 213-bp fragments (bottom panel). L, 100-bp ladder. (C) The OCD-associated region at 9p24 containing SLC1A1. The 39-kb region identified in early-onset OCD cohorts is represented by the red line; SLC1A1 coding regions are represented by gray bars; untranslated regions are represented by vertical white bars; blue shading indicates a significant association with OCD (P < 0.05); red shading indicates an experimentally validated loss-of-function mutation; stacked boxes represent SNPs; blue triangles represent markers linked to OCD; connected blue circles indicate haplotype blocks significantly associated with OCD (P < 0.05); lower boxes indicate missense mutations (c.490A > G [T164A, ref. 40]), c.1184–1186delTCA [I395del], c.1333C > T [R445W]); red arrow indicates an association with OCD-like features; and coordinates represent telomeric distance (in Mb). Sex-specific association with OCD is indicated (male [M] or female [F]). Roman numerals correlate with individual OCD studies: i (20); ii (21); iii (26); iv (27); v (25); vi (22); vii (23); and viii (24).

(A) Highly conserved regions in orthologs of SLC1A1 (top group) and SLC1 family members (middle group) and the SLC1A1 consensus sequence (bottom). Residues I395 and R445, deleted and mutated, respectively, in DA pedigrees are indicated. Residues known to be functionally important in SLC1A1, aspartate 444 (31) and arginine 447 (30), are indicated (diamonds). Blocks of the same color highlight groups of the same or similar amino acids: yellow, hydrophobic; red, acidic; orange, basic; purple, aromatic; blue, amido; green, hydroxyl; gray, proline; and pink, sulfur containing. (B) Predicted location of mutant residues based on the crystal structure of the prokaryotic glutamate transporter (Glt_Ph). R445 (equivalent to M395 in Glt_Ph) is predicted to be located in transmembrane 8, and I395 (equivalent to I339 in Glt_Ph) is in hairpin loop 2 (HP2) near the hinge region. Blue spheres indicate sodium ions; Asp represents the aspartate substrate.

(A) Representative oocytes expressing WT, R445W, or I395del cRNA or noninjected oocytes (control) were clamped at –60 mV and perfused with ND96 buffer containing 100 μM (for WT and R445W) or 1 mM (for I395del and control) l-glutamate. Black scale bars represent the time (s, x axis) that l-glutamate was applied and the current size (nA, y axis). Scale is shown for time versus current. (B) The l-glutamate dose response for WT (filled circles) and R445W (open circles). Current (I_norm) was normalized to the maximal current, defined as unit I_max (WT, K_0.5 = 30 ± 4 μM and I_max = 424 ± 13 nA; R445W, K_0.5 = 2 ± 0.3 μM and I_max 53 ± 7 nA). (C) Radiolabeled l-glutamate uptake at K_0.5. (D) The l-cysteine dose response for WT (filled circles) and R445W (open circles). Current was normalized to the maximal current (WT, K_0.5 = 115 ± 13 μM and I_max = 590 ± 19 nA; R445W, K_0.5 = 3.3 ± 0.1 μM and I_max = 54 ± 20 nA). (E) Radiolabeled l-cysteine uptake at K_0.5. ³H-l-glutamate and ³⁵S-l-cysteine uptake in noninjected oocytes was subtracted from the data presented (C and E, respectively). All data represent the mean ± SEM of at least 3 oocytes (in B–E). (F–H) Representative oocytes expressing EGFP-tagged SLC1A1 cRNA. (F) WT oocytes. (G) R445W oocytes. (H) Noninjected control oocytes. Scale bar: 200 μm.

(A–D) Immunofluorescence of hemagglutinin epitope-tagged WT and mutant cDNAs stably expressed in MDCKII cells (A) SLC1A1 WT, (B) SLC1A1 R445W, and (C) SLC1A1 I395del MDCKII, and (D) empty vector (pcDNA3.1-HA). Scale bar: 10 μm. (E–H) Immunofluorescence of normal adult human kidney, showing SLC1A1 expression in proximal tubules. Kidney sections were stained with an SLC1A1 antibody (red) and the proximal tubule marker LTA (green). Nuclei were stained with DAPI (blue). (E) SLC1A1 expression on the apical membrane of kidney tubules. An example of an S1 segment (arrow) emerging from the glomerulus (g) is indicated. (F) LTA staining identifying the apical membrane of proximal tubules. (G) Colocalization (yellow) of SLC1A1 and LTA on the apical membrane of S1 segments of the proximal tubule (confocal images in E and F were merged). (H) Peptide-blocked control. A glomerulus is indicated. Scale bar: 30 μm.

A nephron is depicted (inset), showing the glomerulus, proximal convoluted tubule (PCT), proximal straight tubule (PST), distal convoluted tubule (DCT), and collecting duct (CD). A cross-section of the proximal convoluted tubule (white square indicated with an arrow) is represented in the main diagram. Four of the aminoacidurias, including DA, iminoglycinuria, Hartnup disorder, and cystinuria manifest at the apical surface of the renal tubule, while lysinuric protein intolerance manifests at the basolateral surface. Mutations in the high-affinity glutamate and aspartate transporter SLC1A1, responsible for DA, were identified in this study. Iminoglycinuria results from complete inactivation of SLC36A2, a proline and glycine transporter, or from additional modifying mutations in the high-affinity proline transporter SLC6A20 when SLC36A2 is not completely inactivated (18). Mutations in the neutral amino acid transporter, SLC6A19, are responsible for Hartnup disorder (16, 17). The neutral amino acid transport defect can also be exacerbated by a kidney-specific loss of heterodimerization of mutant SLC6A19 with TMEM27 (44). Cystinuria has a heterogeneous phenotype and arises from mutations in individual or both subunits of the disulfide bridge-linked heterodimer comprising the type II membrane protein SLC3A1 (12) and the cystine and basic amino acid transporter SLC7A9 (13). Lysinuric protein intolerance (14, 15) results from mutations in the basolaterally expressed basic amino acid transporter SLC7A7, which forms a disulfide bridge-linked heterodimer with type II membrane protein SLC3A2. The major transporters involved in each amino­aciduria are in bold. For a detailed review of renal epithelial amino acid transport systems and their involvement in disease see Bröer (45).