Renal salt loss in Bartter's syndrome is caused by impaired transepithelial transport in the loop of Henle. Sodium chloride is taken up apically by the combined activity of NKCC2 (Na+-K--2Cl- cotransporters) and ROMK potassium channels. Chloride ions exit from the cell through basolateral ClC-Kb chloride channels. Mutations in the three corresponding genes have been identified that correspond to Bartter's syndrome types 1-3. The gene encoding the integral membrane protein barttin is mutated in a form of Bartter's syndrome that is associated with congenital deafness and renal failure. Here we show that barttin acts as an essential beta-subunit for ClC-Ka and ClC-Kb chloride channels, with which it colocalizes in basolateral membranes of renal tubules and of potassium-secreting epithelia of the inner ear. Disease-causing mutations in either ClC-Kb or barttin compromise currents through heteromeric channels. Currents can be stimulated further by mutating a proline-tyrosine (PY) motif on barttin. This work describes the first known beta-subunit for CLC chloride channels and reveals that heteromers formed by ClC-K and barttin are crucial for renal salt reabsorption and potassium recycling in the inner ear.

Molecular biology entered the field of mammalian amino acid transporters in 1990-1991 with the cloning of the first GABA and cationic amino acid transporters. Since then, cDNA have been isolated for more than 20 mammalian amino acid transporters. All of them belong to four protein families. Here we describe the tissue expression, transport characteristics, structure-function relationship, and the putative physiological roles of these transporters. Wherever possible, the ascription of these transporters to known amino acid transport systems is suggested. Significant contributions have been made to the molecular biology of amino acid transport in mammals in the last 3 years, such as the construction of knockouts for the CAT-1 cationic amino acid transporter and the EAAT2 and EAAT3 glutamate transporters, as well as a growing number of studies aimed to elucidate the structure-function relationship of the amino acid transporter. In addition, the first gene (rBAT) responsible for an inherited disease of amino acid transport (cystinuria) has been identified. Identifying the molecular structure of amino acid transport systems of high physiological relevance (e.g., system A, L, N, and x(c)- and of the genes responsible for other aminoacidurias as well as revealing the key molecular mechanisms of the amino acid transporters are the main challenges of the future in this field.

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney >>> placenta > > brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or ϳ286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells.Last year, three amino acid transporter cDNAs (LAT-1, y ϩ LAT-1, and y ϩ LAT-2) 1 were identified as subunits of the heavy chain of the cell surface antigen 4F2 (4F2hc, also named CD98) (1-3). These subunits co-express amino acid transport activity with 4F2hc in oocytes (i.e. system L for LAT-1, and system y ϩ L for y ϩ LAT-1 and y ϩ LAT-2) (1-4). The role of this family of proteins in amino acid transport has recently been demonstrated by the fact that mutations in the y ϩ LAT-1 gene cause lysinuric protein intolerance, an inherited amino aciduria due to a defective renal reabsorption mechanism of dibasic amino acids (5, 6). The structural and functional similarities between 4F2hc and its homologous protein rBAT suggest that a member of this family of subunits might be the subunit of rBAT needed to fully express the amino acid transport system b o,ϩ activity (reviewed in Refs. 7 and 8). After the identification of rBAT as the Type I cystinuria gene (9), this subunit is a good candidate for non-Type I cystinuria (7). A search throughout gene data bases suggests that there may be as many as four new human members of the family of subunits of 4F2hc and rBAT.Kanai and co-workers (1) identified rat LAT-1 (also known as TA1) by co-expression cloning with 4F2hc in oocytes. The coexpressed transport activity shows clear characteristic...

We have identified a new human cDNA (y ؉ L amino acid transporter-1 (y ؉ LAT-1)) that induces system y ؉ L transport activity with 4F2hc (the surface antigen 4F2 heavy chain) in oocytes. Human y ؉ LAT-1 is a new member of a family of polytopic transmembrane proteins that are homologous to the yeast high affinity methionine permease MUP1. Other members of this family, the Xenopus laevis IU12 and the human KIAA0245 cDNAs, also co-express amino acid transport activity with 4F2hc in oocytes, with characteristics that are compatible with those of systems L and y ؉ L, respectively. y ؉ LAT-1 protein forms a Ϸ135-kDa, disulfide bond-dependent heterodimer with 4F2hc in oocytes, which upon reduction results in two protein bands of Ϸ85 kDa (i.e. 4F2hc) and Ϸ40 kDa (y ؉ LAT-1). Mutation of the human 4F2hc residue cysteine 109 (Cys-109) to serine abolishes the formation of this heterodimer and drastically reduces the coexpressed transport activity. These data suggest that y ؉ LAT-1 and other members of this family are different 4F2 light chain subunits, which associated with 4F2hc, constitute different amino acid transporters. Human y ؉ LAT-1 mRNA is expressed in kidney > > peripheral blood leukocytes > > lung > placenta ‫؍‬ spleen > small intestine. The human y ؉ LAT-1 gene localizes at chromosome 14q11.2 (17cR Ϸ 374 kb from D14S1350), within the lysinuric protein intolerance (LPI) locus (Lauteala, T., Sistonen, P., Savontaus, M. L., Mykkanen, J., Simell, J., Lukkarinen, M., Simmell, O., and Aula, P. (1997) Am. J. Hum. Genet. 60, 1479 -1486). LPI is an inherited autosomal disease characterized by a defective dibasic amino acid transport in kidney, intestine, and other tissues. The pattern of expression of human y ؉ LAT-1, its coexpressed transport activity with 4F2hc, and its chromosomal location within the LPI locus, suggest y ؉ LAT-1 as a candidate gene for LPI.rBAT and 4F2hc are homologous proteins that induce amino acid transport in Xenopus oocytes (1, 2). These two proteins are slightly hydrophobic, which prompted the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transporter. This has been supported by several indirect observations: (i) rBAT and 4F2hc are involved in the induction of several activities in Xenopus oocytes (3-6); (ii) these two proteins can be immunodetected or immunoprecipitated as complexes of Ϸ125 kDa in the absence of reducing agents and as two proteins of Ϸ85 kDa (4F2hc or rBAT) and Ϸ40 kDa in the presence of reducing agents (7-9); and (iii) in oocytes, there is a dissociation between the expression of 4F2hc and rBAT at the plasma membrane and the induction of system y ϩ L and b 0,ϩ activity, respectively, indicating that this expression is limited by an endogenous factor (10, 11). We have recently provided new evidence that the amino acid transport system y ϩ L has a heterodimeric structure (11). Thus, we have shown that the y ϩ L activity induced in oocytes by a cysteineless mutant of human 4F2hc is also inactivated by membraneimpermeant thiol-specific ...

SummaryIon fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl− channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.Video Abstract