1,721,007 research outputs found
Passive water permeability of some wild type and mutagenized amino acid cotransporters of the SLC6/NSS family expressed in Xenopus laevis oocytes
No full textIn 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
Structure and function of the K+ -dependent amino acid cotransporter KAAT1 cloned from Manduca sexta midgut
No full textChloride interaction in the SLC6 amino acid cotransporter KAAT1
No full textThe SLC6 family of solute transporters groups eukaryotic, Cl--dependent proteins as the serotonin transporter SERT (1) and the GABA transporter GAT1 (2) and prokaryotic, Cl--independent proteins, as the family model LeuT (3, 4). The main role of the anion in the transport cycle appears to be related to the neutralization of the positive charges of sodium ions transported with the substrate. KAAT1 is a lepidopteran SLC6 amino acid cotransporter, activated by Na+ and K+, but characterized by a weak chloride dependence (5, 6). In recent years, taking advantage of the special features of KAAT1, we have investigated the structural/functional relationships within the SLC6 family. By site-directed mutagenesis and functional expression in Xenopus laevis oocytes, we have identified residues involved in Na+ and K+ interaction and in amino acid translocation (7,8,9). The aim of this study has been the identification of the molecular determinants of chloride interaction of KAAT1 to obtain insights in the transport mechanism of SLC6 members. Comparison of KAAT1 sequence with SERT, GAT1 and LeuT has revealed some differences in residues forming the putative anion binding site but, among theme, only T339 seemed to be relevant for chloride dependence: T339S and T339E mutant transport activity became indeed almost completely Cl--dependent. The mutation of a further residue (T67), conserved only in KAAT1 and in the other weakly Cl--dependent transporter of the family CAATCH1, affected KAAT1 activity: T67Y mutant was fully chloride independent whereas T67S and T67A showed an enhancement in chloride dependence. In order to confirm the role of T67, we built the reciprocal mutant of KAAT1 T67Y in GAT1 transporter: Y60T, interestingly, showed a reduced chloride dependence compared to the wt. These data suggest that T67 and T339 influence KAAT1 interaction with chloride.
1. Forrest L.R. et al., Proc Natl Acad Sci U S A. 2007; 104:12761-6.
2. Zomot E. et al., Nature. 2007 Oct 11; 449: 726-30.
3. Yamashita A. et al., Nature. 2005; 437: 215-23
4. Ben-Yona A. et al., J Biol Chem. 2011; 286: 2826-33
5. Castagna M. et al., Proc Natl Acad Sci U S A.; 95: 5395-400
6. Bettè S. et al., Channels (Austin). 2008; 2: 358-62
7. Mari et al., Cell. Mol. Life Sci. 2004, 61: 243-56.
8. Miszner et al., J. Physiol. 2007, 58: 1899-1913.
9. Castagna et al., Am. J. Physiol. Cell. Physiol. 2007, 293: C1286-C129
Extracellular ionic concentrations in Bombyx mori L. central nervous system
No full textAim of this study was to determine the ionic concentration gradients at the cellular membrane level in the central nervous system of an insect where the haemolymphatic sodium concentration relative to that of potassium is too low to allow action potential generation. The intracellular ionic concentrations determined by electrophysiological experiments performed at different Na and K concentrations in the perfusing solution, the Na and K concentrations in the nerve cord tissue water, and the extracellular spaces observed by electron microscopy, support the conclusion that the cells in the ventral nerve cord of Bombyx mori function in a medium with a relatively low potassium content and a high sodium concentration. This fluid is in a microenvironment protected by a barrier, the perineurium and associated glial elements, where must be located a regulation mechanism of cation composition. In order to aid the microelectrode penetration the preparations were exposed to Pronase. The Pronase action was investigated by electron microscopy and determinations of ionic concentrations
- …
