Passive water permeability of some wild type and mutagenized amino acid cotransporters of the SLC6/NSS family expressed in Xenopus laevis oocytes.
ABSTRACT In this paper passive water movement across the cell membrane mediated by wild type and mutagenized cotransporters was investigated. We evaluated water movement and, in parallel, amino acid uptake induced by some members of the SLC6/NSS family belonging to different kingdoms, namely the rat GABA transporter GAT1, the insect amino acid transporters KAAT1 and CAATCH1 and the bacterial leucine transporter LeuT, whose structure was recently solved. We also tested whether mutated proteins in which the solute translocation mechanism is altered or even abolished were able to induce water movement across cell membrane. The proteins of interest were expressed in Xenopus laevis oocytes and osmotic water permeabilities were estimated from the rate of cell volume change induced by an osmotic gradient in the absence of cotransported solutes. Under osmotic stress all the studied wild type amino acid cotransporters increased the water permeability of the membrane. The GABA transport inhibitor SKF 89976A inhibited both GABA transport and water movement induced by the expression of GAT1. Interestingly, the capacity of mutant proteins to induce water movement was not predictable on the basis of their substrate transport ability. In particular the GAT1 mutant Q291N, void of any transport activity, induced a water permeability similar to that induced by the wt protein. The KAAT1 mutant T339C, which showed a higher transport activity, induced a water permeability not significantly different from the wild type transporter. Interestingly, the bacterial leucine cotransporter LeuT, whose binding site for leucine and Na(+) is void of water, induced water movement through the plasma membrane.
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ABSTRACT: 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.Cellular and Molecular Life Sciences CMLS 02/2004; 61(2):243-56. · 5.62 Impact Factor
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ABSTRACT: Water traverses the plasma membranes of some eukaryotic cells faster than can be explained by the water permeability of their lipid bilayers. This has led to a search for a water channel. Our previous work identified glucose transporters as candidates for such a channel. We report here that Xenopus laevis oocytes injected with mRNA encoding the brain/Hep G2, adult skeletal muscle/adipocyte, or liver forms of the glucose transporter exhibit an osmotic water permeability of their plasma membranes larger than that of untreated oocytes. The osmotic water permeability component attributable to glucose transporters increased an average of 4.8-fold in the injected oocytes. These studies provide direct evidence that the facilitative, sodium-independent mammalian glucose transporters serve as membrane water channels.Proceedings of the National Academy of Sciences 05/1990; 87(8):3244-7. · 9.74 Impact Factor
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ABSTRACT: We have investigated the functional role of Cl− in the human Na+/Cl−/γ-aminobutyric acid (GABA) and Na+/glucose cotransporters (GAT1 and SGLT1, respectively) expressed in Xenopus laevis oocytes. Substrate-evoked steady-state inward currents were examined in the presence and absence of external Cl−. Replacement of Cl− by gluconate or 2-(N-morpholino)ethanesulfonic acid decreased the apparent affinity of GAT1 and SGLT1 for Na+ and the organic substrate. In the absence of substrate, GAT1 and SGLT1 exhibited charge movements that manifested as pre-steady-state current transients. Removal of Cl− shifted the voltage dependence of charge movements to more negative potentials, with apparent affinity constants (K 0.5) for Cl− of 21 and 115 mm for SGLT1 and GAT1, respectively. The maximum charge moved and the apparent valence were not altered. GAT1 stoichiometry was determined by measuring GABA-evoked currents and the unidirectional influx of 36Cl−,22Na+, or [3H]GABA. Uptake of each GABA molecule was accompanied by inward movement of 2 positive charges, which was entirely accounted for by the influx of Na+ in the presence or absence of Cl−. Thus, the GAT1 stoichiometry was 2Na+:1GABA. However, Cl− was transported by GAT1 because the inward movement of 2 positive charges was accompanied by the influx of one Cl− ion, suggesting unidirectional influx of 2Na+:1Cl−:1GABA per transport cycle. Activation of forward Na+/Cl−/GABA transport evoked 36Cl− efflux and was blocked by the inhibitor SKF 89976A. These data suggest a Cl−/Cl− exchange mechanism during the GAT1 transport cycle. In contrast, Cl− was not transported by SGLT1. Thus, in both GAT1 and SGLT1, Cl− modulates the kinetics of cotransport by altering Na+ affinity, but does not contribute to net charge transported per transport cycle. We conclude that Cl− dependence per se is not a useful criterion to classify Na+ cotransporters.Journal of Biological Chemistry 11/2000; 275(48):37414-37422. · 4.65 Impact Factor