Molecular cloning of the mouse IMINO system: an Na+- and CI--dependent proline transporter

Neurotransmitter transporters of the SLC6 family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. Here we demonstrate that the mouse homologue of slc6a20 has all properties of th

Molecular Cloning of Mouse Amino Acid Transport System B 0 , a Neutral Amino Acid Transporter Related to Hartnup Disorder

Resorption of amino acids in kidney and intestine is mediated by transporters, which prefer groups of amino acids with similar physico-chemical properties. It is generally assumed that most neutral amino acids are transported across the apical membrane of epithelial cells by system B 0. Here we have characterized a novel member of the Na +-dependent neurotransmitter transporter family (B0AT1) isolated from mouse kidney, which shows all properties of system B0. Flux experiments showed that the transporter is Na+-dependent, electrogenic, and actively transports most neutral amino acids but not anionic or cationic amino acids. Superfusion of mB0AT1-expressing oocytes with neutral amino acids generated inward currents, which were proportional to the fluxes observed with labeled amino acids. In situ hybridization showed strong expression in intestinal microvilli and in the proximal tubule of the kidney. Expression of mouse B0AT1 was restricted to kidney, intestine, and skin. It is generally assumed that mutations of the system B 0 transporter underlie autosomal recessive Hartnup disorder. In support of this notion mB0AT1 is located on mouse chromosome 13 in a region syntenic to human chromosome 5p15, the locus of Hartnup disorder. Thus, the human homologue of this transporter is an excellent functional and positional candidate for Hartnup disorder

The orphan transporter v7-3 (slc6a15) is a Na(+)-dependent neutral amino acid transporter (B(0)AT2)

Transporters of the SLC6 (solute carrier 6) family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. In the present study, we demonstrate that mouse v7-3 (slc6a15) encodes a transporter for neutral amino acids. The transporter is functionally and sequence related to B(0)AT1 (slc6a19) and was hence named B(0)AT2. Leucine, isoleucine, valine, proline and methionine were recognized by the transporter, with values of K(0.5) (half-saturation constant) ranging from 40 to 200 μM. Alanine, glutamine and phenylalanine were low-affinity substrates of the transporter, with K(0.5) values in the millimolar range. Transport of neutral amino acids via B(0)AT2 was Na(+)-dependent, Cl(−)-independent and electrogenic. Superfusion of mouse B(0)AT2-expressing oocytes with amino acid substrates generated robust inward currents. Na(+)-activation kinetics of proline transport and uptake under voltage clamp suggested a 1:1 Na(+)/amino acid co-transport stoichiometry. Susbtrate and co-substrate influenced each other's K(0.5) values, suggesting that they share the same binding site. A mouse B(0)AT2-like transport activity was detected in synaptosomes and cultured neurons. A potential role of B(0)AT2 in transporting neurotransmitter precursors and neuromodulators is proposed

Further Evidence for Allelic Heterogeneity in Hartnup Disorder

Hartnup disorder is an autosomal recessive impairment of amino acid transport in kidney and intestine. Mutations in SLC6A19 have been shown to cosegregate with the disease in the predicted recessive manner; however, in two previous studies (Seow et al., Nat Genet 2004;36:1003-1007; Kleta et al., Nat Genet 2004;36:999-1002), not all causative alleles were identified in all affected individuals, raising the possibility that other genes may contribute to Hartnup disorder. We have now investigated six newly acquired families of Australian and Canadian (Province of Quebec) origin and resequenced the entire coding region of SLC6A19 in families with only a single disease allele identified. We also studied one American family in whom no mutations had been identified in a previous study (Kleta et al., Nat Genet 2004;36:999-1002). We have identified seven novel mutations in SLC6A19 that show functional obliteration of the protein in vitro, explaining Hartnup disorder in all reported families so far. We demonstrate that Hartnup disorder is allelically heterogeneous with two mutated SLC6A19 alleles, whether identical or not, necessary for manifestation of the characteristic aminoaciduria in affected individuals. This study resolves the previous hypothesis that other genes contribute to the Hartnup phenotype

Iminoglycinuria and hyperglycinuria are discrete human phenotypes resulting from complex mutations in proline and glycine transporters

Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes

Human retinal pigment epithelium cells can be imaged in vivo with a novel adaptive optics camera using transscleral illumination

Histopathology studies described morphological changes of the retinal pigment epithelium (RPE) specific to the onset and progression of retinal diseases in human eyes. However to date, no valuable imaging tool is used in the clinic to image RPE cells. Transscleral Optical Imaging (TOI) uses an oblique illumination of the fundus combined with adaptive optics to provide cell-resolution images of the retinal layers up to the RPE, in vivo. A prospective study was carried out to assess safety and repeatability of TOI technology and characterize healthy RPE cells

Human retinal pigment epithelium cells can be imaged in vivo with a novel adaptive optics camera using transscleral illumination

Histopathology studies described morphological changes of the retinal pigment epithelium (RPE) specific to the onset and progression of retinal diseases in human eyes. However to date, no valuable imaging tool is used in the clinic to image RPE cells. Transscleral Optical Imaging (TOI) uses an oblique illumination of the fundus combined with adaptive optics to provide cell-resolution images of the retinal layers up to the RPE, in vivo. A prospective study was carried out to assess safety and repeatability of TOI technology and characterize healthy RPE cells

Characterization of mouse amino acid transporter B(0)AT1 (slc6a19)

The mechanism of the mouse (m)B(0)AT1 (slc6a19) transporter was studied in detail using two electrode voltage-clamp techniques and tracer studies in the Xenopus oocyte expression system. All neutral amino acids induced inward currents at physiological potentials, but large neutral non-aromatic amino acids were the preferred substrates of mB(0)AT1. Substrates were transported with K(0.5) values ranging from approx. 1 mM to approx. 10 mM. The transporter mediates Na(+)–amino acid co-transport with a stoichiometry of 1:1. No other ions were involved in the transport mechanism. An increase in the extracellular Na(+) concentration reduced the K(0.5) for leucine, and vice versa. Moreover, the K(0.5) values and V(max) values of both substrates varied with the membrane potential. As a result, K(0.5) and V(max) values are a complex function of the concentration of substrate and co-substrate and the membrane potential. A model is presented assuming random binding order and a positive charge associated with the ternary [Na(+)–substrate–transporter] complex, which is consistent with the experimental data