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...

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, KAATI 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(Il)(l,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.

Xenopus laevis oocytes are an interesting model for the study of many developmental mechanisms because of their dimensions and the ease with which they can be manipulated. In addition, they are widely employed systems for the expression and functional study of heterologous proteins, which can be expressed with high efficiency on their plasma membrane. Here we applied atomic force microscopy (AFM) to the study of the plasma membrane of X. laevis oocytes. In particular, we developed and optimized a new sample preparation protocol, based on the purification of plasma membranes by ultracentrifugation on a sucrose gradient, to perform a high-resolution AFM imaging of X. laevis oocyte plasma membrane in physiological-like conditions. Reproducible AFM topographs allowed visualization and dimensional characterization of membrane patches, whose height corresponds to a single lipid bilayer, as well as the presence of nanometer structures embedded in the plasma membrane and identified as native membrane proteins. The described method appears to be an applicable tool for performing high-resolution AFM imaging of X. laevis oocyte plasma membrane in a physiological-like environment, thus opening promising perspectives for studying in situ cloned membrane proteins of relevant biomedical/pharmacological interest expressed in this biological system.

Intermittent contact mode atomic force microscopy (AFM) was used to visualize the native plasma membrane of Xenopus laevis oocytes. Oocyte membranes were purified via ultracentrifugation on a sucrose gradient and adsorbed on mica leaves. AFM topographs and the corresponding phase images allowed for visualization and identification of both oocyte plasma membrane patches and pure lipid bilayer regions with a height of about 5 nm within membrane patches. The quantitative analysis showed a normal distribution for the lateral dimension and height of the protein complexes centered on 16.7 +/- 0.2 nm (mean +/- SE, n = 263) and 5.4 +/- 0.1 nm (n = 262), respectively. The phase signal, providing material-dependent information, allowed for the recognition of structural features observed in AFM topographs.

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