† These authors contributed equally to the study.The glutamate transporter excitatory amino acid carrier (EAAC1/EAAT3) mediates the absorption of dicarboxylic amino acids in epithelial cells as well as the uptake of glutamate from the synaptic cleft. Its cell-surface density is regulated by interaction with accessory proteins which remain to be identified. We detected a consensus sequence for interaction with post-synaptic density- 95/Discs large/Zonula occludens (PDZ) proteins (-SQF) and a tyrosine-based internalization signal (-YVNG-) in the C-terminus of EAAC1, and investigated their role in the transporter localization.We demonstrated that PDZ interactions are required for the efficient delivery to and the retention in the plasma membrane of EAAC1 and we identified PDZK1/NHERF3 (Na+/H+-exchanger regulatory factor 3) as a novel EAAC1 interacting protein. Expression of PDZK1 in Madin-Darby canine kidney (MDCK) cells tethered EAAC1 to filopodia and increased its surface activity. Removal of the PDZ-target motif promoted the EAAC1 binding to α-adaptin and clathrin and the transporter internalization in endocytic/degradative compartments. This defect was largely prevented by hypertonic treatment or overexpression of the dominant-negative μ2-W421A-subunit of AP-2 clathrin-adaptor. The rate of transporter endocytosis was attenuated following tyrosine mutagenesis in the internalization signal, thus indicating that this motif can regulate the transporter endocytosis.We suggest that EAAC1 density is controlled by balanced interactions with PDZK1 and adaptor protein 2 (AP2): the former promotes the transporter expression at the cell surface, and the latter mediates its constitutive endocytosis.

Structural domains involved in substrate selectivity in two neutral amino acid transporters. Am J Physiol Cell Physiol 287: C754 -C761, 2004. First published May 12, 2004 10.1152/ajpcell.00016.2004The ability of the two highly homologous Na ϩ /Cl Ϫ -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 transportassociated 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 measurements. Initially, we exchanged the first three domains, obtaining the chimeras C3K9 and K3C9 (where numbers indicate the transmembrane domains and letters represent the original proteins), which showed electrophysiological and [ 3 H]amino acid uptake characteristics resembling those of KAAT1 and CAATCH1, respectively. Subsequent substitution of the last four domains in C3K9 and K3C9 gave the proteins C3K5C4 and K3C5K4, which showed the same behavior as KAAT1 and CAATCH1 in electrophysiological and transport determinations. These results suggest that in KAAT1 and CAATCH1, only the central transmembrane domains (from 4 to 8) of the protein are responsible for substrate selectivity. structure and function; Manduca sexta COMPARING SEQUENCES AND FUNCTIONS of related proteins can give some insights on the physiological role of particular domains. In the present study, we analyzed two amino acid transporters cloned from the Manduca sexta larva, the tobacco hornworm, which is a lepidopteran of great agroeconomical importance and is one of the best-studied insects as a result of the ease in rearing it on an artificial diet. The intestine of this larva has been the source for the cloning of the two transporters used in the present work: K ϩ -coupled amino acid transporter 1 (KAAT1) (6) and cation-amino acid transporter/channel 1 (CAATCH1) (9). The two proteins, of 634 and 633 amino acids, respectively, are very similar to each other (90% amino acid identity), and their 35-45% identity with the members of the Na ϩ /Cl Ϫ -dependent neurotransmitter transporters allows them to be included in this important superfamily (6, 9). Accordingly, their hydropathicity profiles suggest the presence of 12 transmembrane domains with cytosolic carboxy and amino termini. The two transporters share the peculiar property of being able to utilize the K ϩ gradient to en...

To investigate the peculiar ionic specificity of KAAT1, an Na+- and K+-coupled amino acid cotransporter from Lepidoptera, a detailed analysis of membrane topology predictions was performed, together with sequence comparison with strictly Na+-dependent mammalian cotransporters from the same family. The analysis identified aspartate 338, a residue present also in the other cotransporter accepting K+ (CAATCH1), but absent in most mammalian transporters that have, instead, an asparagine in the corresponding position. Mutation of D338 in KAAT1 led either to non-functional transporters (D338G, D338C), or to an altered ionic selectivity (D338E, D338N), observable in uptake experiments and in electrophysiological properties. In particular, in D338E, the transport activity, while persisting in the presence of Na+, appeared to be completely abolished in the presence of K+. D338E also showed uncoupling between transport-associated current and uptake. The opposite mutation in the gamma-aminobutyric acid transporter rGAT-1 (N327D) resulted in complete loss of function. In conclusion, aspartate 338 in KAAT1 appears to be important in allowing K+, in addition to Na+, to drive the transport mechanism, although other residues in different parts of the protein may also play a role in the complete determination of ionic selectivity.

Abstract. We investigated the role of the Q291 glutamine residue in the functioning of the rat g-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transportcoupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wildtype and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhyd- Cell. Mol. Life Sci. 63 (2006) 0100-0111 1420-682X/06/010100-12 DOI 10.1007/s00018-005-5512-6 © Birkhäuser Verlag, Basel, 2006 ryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter. Key words. Neurotransmitter transporter; GAT-1; electrophysiology; site-directed mutagenesis; Xenopus laevis oocyte; structure-function relationship.g-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in mammalian brain, and the GABA transporter GAT-1 is an integral membrane protein responsible for the reuptake of GABA from the synaptic cleft during neurotransmission. GAT-1 was the first cloned member of a large family of homologous proteins, the Na + /Cl --dependent neurotransmitter transporters [1], which includes transporters for other neurotransmitters such as norepinephrine, dopamine, serotonin and glycine, as well as for a number of other substrates that share the common property of being amino acids or amino acid derivatives [2]. These membrane proteins are predicted to have 12 transmembrane-spanning domains (TMDs) linked by hydrophilic loops with the NH 2 and COOH terminals inside the cell, a topological model that has been confirmed for the serotonin transporter by means of sitedirected chemical labeling [3]. GAT-1 mediates the electrogenic reuptake of GABA in the presence of sodium and chloride ions by means of a mechanism that has been extensively studied [4][5][6][7][8][9][10][11][12]. Many attempts have been made to determine which of the transporter amino acid residues are involved in the interaction with GABA and with Na + and Cl -ions, and which participate in the conformational changes associated with translocation [13][14][15]. As GABA is a zwitterionic molecule and its cosubstrates are charged species, the first studies focused on the charged and conserved residues predicted to be located in (or adjacent to) the transmembrane (TM) domains. This allowed the identification of * Corresponding author. Cellular and Molecular Life Sciences Research ArticleRole of the conserved glutamine 291 in the r...

We have combined structural and functional approaches to investigate the role of oligomerization in the operation of the GABA transporter rGAT1. Xenopus laevis oocytes were induced to express, either separately or simultaneously, the wild-type form of rGAT1 and a mutated (Y140W) form, unable to translocate GABA and to generate transport currents, although its intramembrane charge movement properties are only slightly affected. These characteristics, together with the insensitivity of Y140W to the blocking action of SKF89976A, were used to study the possible functional interaction of the two forms in an heteromeric structure. The electrophysiological data from oocytes coexpressing wild-type and Y140W rGAT1 were consistent with a completely independent activity of the two forms. Oligomerization was also studied by fluorescence resonance energy transfer (FRET) in tsA201 cells expressing the transporters fused with cyan and yellow fluorescent proteins (ECFP and EYFP). All combinations tested (WT-ECFP/WTEYFP, Y140W-ECFP/Y140W-EYFP and WT-ECFP/ Y140W-EYFP) were able to give rise to FRET, confirming the formation of homo- as well as heterooligomers. We conclude that, although rGAT1 undergoes structural oligomerization, each monomer operates independently.