S. A. Mari

Role of the conserved glutamine 291 in the rat γ-aminobutyric acid transporter rGAT-1

Abstract.We investigated the role of the Q291 glutamine residue in the functioning of the rat γ-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wild-type 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 sulfhydryl 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.

Aspartate 338 contributes to the cationic specificity and to driver-amino acid coupling in the insect cotransporter KAAT1

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 γ-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.

Flavone and xanthone derivatives related to fluoroquinolones

A number of flavone and xanthone derivatives bearing some characteristic features of fluoroquinolones such as the fluorine atom and an ortho piperazine ring are described. The new compounds have been tested for possible cytotoxic and antimicrobial activities. Cytotoxicity of both groups of compounds is rather poor, while the antibacterial activity is restricted to xanthones.

Atomic force microscopy imaging of actin cortical cytoskeleton of Xenopus laevis oocyte

In this study we report an atomic force microscopy (AFM) investigation of the actin cortical cytoskeleton of Xenopus laevis oocytes. Samples consisted of inside‐out orientated plasma membrane patches of X. laevis oocytes with overhanging cytoplasmic material. They were spread on a freshly cleaved mica surface, subsequently treated with Triton X‐100 detergent and chemically fixed. The presence of actin fibres in oocyte patches was proved by fluorescence microscopy imaging. Contact mode AFM imaging was performed in air in constant force conditions. Reproducible high‐resolution AFM images of a filamentous structure were obtained. The filamentous structure was identified as an actin cortical cytoskeleton, investigating its disaggregation induced by cytochalasin D treatment. The thinnest fibres showed a height of 7 nm in accordance with the diameter of a single actin microfilament. The results suggest that AFM imaging can be used for the high‐resolution study of the actin cortical cytoskeleton of the X. laevis oocyte and its modifications mediated by the action of drugs and toxins.

Atomic force microscopy imaging of Xenopus laevis oocyte plasma membrane purified by ultracentrifugation.

Atomic force microscopy (AFM) was used to investigate the native plasma membrane of Xenopus laevis (X. laevis) oocyte purified by means of ultracentrifugation on sucrose gradient and subsequently adsorbed on mica leaves through a physisorption process. Reproducible AFM topography images were collected, analyzed, and compared. AFM images showed the presence of large single or double bilayer membrane sheets covered with protein complexes. The lateral dimension and height of protein complexes imaged in air showed a normal distribution centred on 15.4 ± 0.4 nm (mean ± SE; n = 59) and 3.9 ± 0.2 nm (mean ± SE; n = 57), respectively. A density of about 270 protein complexes per square micron was calculated. Less frequently, ordered nanometer domains with densely packed protein complexes arranged in hexagonal patterns were also visualized in AFM images, confirming previously published data. Their lateral dimension and height showed a normal distribution centred on 23.0 ± 0.4 nm (mean ± SE; n = 42) and 1.5 ± 0.6 nm (mean ± SE; n = 90), respectively. A density of about 870 protein complexes per square micrometer was calculated. Advantages and drawbacks of this new sample preparation for AFM imaging are discussed. Microsc. Res. Tech., 2008.