K+ amino acid transporter KAAT1 mutant Y147F has increased transport activity and altered substrate selectivity (original) (raw)
Related papers
Cloning and characterization of a potassium-coupled amino acid transporter
Proceedings of the National Academy of Sciences, 1998
Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na ؉ or H ؉ gradients. The present work extends the Na ؉ and H ؉ dogma by including the H ؉ and K ؉ paradigm. Lepidopteran insect larvae have a high K ؉ and a low Na ؉ content, and their midgut cells lack Na ؉ ͞K ؉ ATPase. Instead, an H ؉ translocating, vacuolar-type ATPase generates a voltage of approximately ؊240 mV across the apical plasma membrane of so-called goblet cells, which drives H ؉ back into the cells in exchange for K ؉ , resulting in net K ؉ secretion into the lumen. The resulting inwardly directed K ؉ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K ؉ -coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes ␣-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K ؉ , Na ؉ , and to a lesser extent Li ؉ were accepted as cotransported ions, but K ؉ is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl ؊ -dependent, and the K ؉ ͞Na ؉ selectivity increased with hyperpolarization of oocytes, ref lecting the increased K ؉ ͞Na ؉ selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na ؉ and Cl ؊ -coupled neurotransmitter transporter family.
The Journal of experimental biology, 1997
In mammalian cells, the uptake of amino acids is mediated by specialized, energy-dependent and passive transporters with overlapping substrate specificities. Most energy-dependent transporters are coupled either to the cotransport of Na+ or Cl- or to the countertransport of K+. Passive transporters are either facilitated transporters or channels. As a prelude to the molecular characterization of the different classes of transporters, we have isolated transporter cDNAs by expression-cloning with Xenopus laevis oocytes and we have characterized the cloned transporters functionally by uptake studies into oocytes using radiolabelled substrates and by electrophysiology to determine substrate-evoked currents. Mammalian transporters investigated include the dibasic and neutral amino acid transport protein D2/NBAT (system b0+) and the Na(+)- and K(+)-dependent neuronal and epithelial high-affinity glutamate transporter EAAC1 (system XAG-). A detailed characterization of these proteins has p...
Role of a conserved glycine triplet in the NSS amino acid transporter KAAT1
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012
K +-coupled amino acid transporter 1 (KAAT1) belongs to the NSS family of solute transporters and it is expressed in the midgut and in salivary glands of Manduca sexta larvae. As more than 80% of family members, KAAT1 shows a stretch of three glycines (G85-G87) that according to the structure of the prototype transporter LeuT, is located close to the access of the permeation pathway. In this work the role of the triplet has been investigated by alanine and cysteine scanning methods in protein heterologously expressed in Xenopus laevis oocytes. All the mutants were functional but the surface expression level was reduced for G85A and G87A mutants and unaffected for G86A mutant. All presented altered amino acid uptake and transport associated currents in the presence of each of the cations (Na + , K + , Li +) that can be exploited by the wt. G87A mutant induced increased uncoupled fluxes in the presence of all the cations. Cross-linking studies, performed by the treatment of cysteine mutants with the oxidative complex Cu(II)(1,10-phenanthroline) 3 , showed that limiting the flexibility of the region by covalent blockage of position 87, causes a significant reduction of amino acid uptake. Na + protected G87C mutant from oxidation, both directly and indirectly. The conserved glycine triplet in KAAT1 plays therefore a complex role that allows initial steps of cation interaction with the transporter.
Journal of Biological Chemistry, 1993
A cDNA has been isolated from human hippocampus that appears to encode a novel Na+-dependent, Cl-independent, neutral amino acid transporter. The putative protein, designated SA", is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na+-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATT's apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines. Carrier-mediated amino acid transport has evolved to maintain transmembrane fluxes of amino acids for cellular nutrition and metabolism. In eukaryotic cells, a large number of distinct amino acid transport systems have been distinguished based on differences in substrate specificity, kinetic properties, and ionic dependence (1, 2). Our knowledge of the structural properties of amino acid transporters is limited. However, recent advances in the cloning of amino acid transporters have begun to provide insights into the molecular basis of carrier-mediated amino acid transport. A cDNA clone, SAAT1, has recently been described that exhibits structural and sequence similarity with
Oligomeric structure of the neutral amino acid transporters KAAT1 and CAATCH1
AJP: Cell Physiology, 2006
The highly homologous neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the Manduca sexta larva, are members of the Na(+)/Cl(-)-dependent transporter family. Recent evidence indicates that transporters of this family form constitutive oligomers. CAATCH1 and KAAT1 give rise to specific kinds of current depending on the transported amino acid, cotransported ion, pH, and membrane voltage. Different substrates induce notably distinct transport-associated currents in the two proteins that represent useful tools in structural-functional studies. To determine whether KAAT1 and CAATCH1 form functional oligomers, we have constructed four concatameric proteins for electrophysiological analysis, consisting of one KAAT1 protein covalently linked to another KAAT1 (K-K concatamer) or to CAATCH1 (K-C concatamer) and vice versa (C-C concatamer and C-K concatamer), and eight constructs where the two transporters were linked to yellow or cyan fluorescent protein in the NH(2) or COOH terminus, to determine the oligomer formation and the relative distance between the different subunits by fluorescence resonance energy transfer (FRET) analysis. Heterologous expression of the concatenated constructs and coinjection of the original proteins in different proportions allowed us to compare the characteristics of the currents to those of the oocytes expressing only the wild-type proteins. All the constructs were fully active, and their electrophysiological behavior was consistent with the activity as monomeric proteins. However, the FRET studies indicate that these transporters form oligomers in agreement with the LeuT(Aa) atomic structure and confirm that the COOH termini of the adjacent subunits are closer than NH(2) termini.
Structural and functional basis of amino acid specificity in the invertebrate cotransporter KAAT1
The Journal of Physiology, 2007
The substrate specificity of KAAT1, a Na +-and K +-dependent neutral amino acid cotransporter cloned from the larva of the invertebrate Manduca sexta and belonging to the SLC6A gene family has been investigated using electrophysiological and radiotracer methods. The specificity of KAAT1 was compared to that of CAATCH1, a strictly related transporter with different amino acid selectivity. Competition experiments between different substrates indicate that both transporters bind leucine more strongly than threonine and proline, the difference between KAAT1 and CAATCH1 residing in the incapacity of the latter to complete the transport cycle in presence of leucine. The behaviour of CAATCH1 is mimicked by the S308T mutant form of KAAT1, constructed on the basis of the atomic structure of a leucine-transporting bacterial member of the family, which indicates the participation of this residue in the leucine-binding site. The reverse mutation T308S in CAATCH1 conferred to this transporter the ability to transport leucine in presence of K +. These results may be interpreted by a kinetic scheme in which, in presence of Na + , the leucine-bound state of the transporter is relatively stable, while in presence of K + and at negative potentials the progression of the leucine-bound form along the cycle is favoured. In this context serine 308 appears to be important in allowing the change to the inward-facing conformation of the transporter following substrate binding, rather than in determining the binding specificity.
Characterization of mouse amino acid transporter B0AT1 (slc6a19)
Biochemical Journal, 2005
The mechanism of the mouse (m)B0AT1 (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 mB0AT1. Substrates were transported with K0.5 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 K0.5 for leucine, and vice versa. Moreover, the K0.5 values and Vmax values of both substrates varied with the membrane potential. As a result, K0.5 and Vmax 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 t...
Structural domains involved in substrate selectivity in two neutral amino acid transporters
American journal of physiology. Cell physiology, 2004
The ability of the two highly homologous Na(+)/Cl(-)-dependent neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva Manduca sexta, to transport different amino acids depends on the cotransported ion, on pH, and on the membrane voltage. Different organic substrates give rise to transport-associated currents with their own characteristics, which are notably distinct between the two proteins. Differences in amplitude, kinetics, and voltage dependence of the transport-associated currents have been observed, as well as different substrate selectivity patterns measured by radioactive amino acid uptake assays. These diversities represent useful tools to investigate the structural determinants involved in the substrate selectivity. To identify these regions, we built four chimeric proteins between the two transporters. These proteins, heterologously expressed in Xenopus laevis oocytes, were analyzed by two-electrode voltage clamp and uptake measu...