36 research outputs found
Ătude fonctionnelle du cotransporteur Na+/glucose (hSGLT1) : courant de fuite, vitesse de cotransport et modĂ©lisation cinĂ©tique
Les rĂ©sultats prĂ©sentĂ©s dans cette thĂšse prĂ©cisent certains aspects de la fonction du cotransporteur Na+/glucose (SGLT1), une protĂ©ine transmembranaire qui utilise le gradient Ă©lectrochimique favorable des ions Na+ afin dâaccumuler le glucose Ă lâintĂ©rieur des cellules Ă©pithĂ©liales de lâintestin grĂȘle et du rein.
Nous avons tout dâabord utilisĂ© lâĂ©lectrophysiologie Ă deux microĂ©lectrodes sur des ovocytes de xĂ©nope afin dâidentifier les ions qui constituaient le courant de fuite de SGLT1, un courant mesurĂ© en absence de glucose qui est dĂ©couplĂ© de la stoechiomĂ©trie stricte de 2 Na+/1 glucose caractĂ©risant le cotransport. Nos rĂ©sultats ont dĂ©montrĂ© que des cations comme le Li+, le K+ et le Cs+, qui nâinteragissent que faiblement avec les sites de liaison de SGLT1 et ne permettent pas les conformations engendrĂ©es par la liaison du Na+, pouvaient nĂ©anmoins gĂ©nĂ©rer un courant de fuite dâamplitude comparable Ă celui mesurĂ© en prĂ©sence de Na+. Ceci suggĂšre que le courant de fuite traverse SGLT1 en utilisant une voie de permĂ©ation diffĂ©rente de celle dĂ©finie par les changements de conformation propres au cotransport Na+/glucose, possiblement similaire Ă celle empruntĂ©e par la permĂ©abilitĂ© Ă lâeau passive. Dans un deuxiĂšme temps, nous avons cherchĂ© Ă estimer la vitesse des cycles de cotransport de SGLT1 Ă lâaide de la technique de la trappe ionique, selon laquelle le large bout dâune Ă©lectrode sĂ©lective (~100 ÎŒm) est pressĂ© contre la membrane plasmique dâun ovocyte et circonscrit ainsi un petit volume de solution extracellulaire que lâon nomme la trappe. Les variations de concentration ionique se produisant dans la trappe en consĂ©quence de lâactivitĂ© de SGLT1 nous ont permis de dĂ©duire que le cotransport Na+/glucose sâeffectuait Ă un rythme dâenviron 13 s-1 lorsque le potentiel membranaire Ă©tait fixĂ© Ă -155 mV. Suite Ă cela, nous nous sommes intĂ©ressĂ©s au dĂ©veloppement dâun modĂšle cinĂ©tique de SGLT1. En se servant de lâalgorithme du recuit simulĂ©, nous avons construit un schĂ©ma cinĂ©tique Ă 7 Ă©tats reproduisant de façon prĂ©cise les courants du cotransporteur
en fonction du Na+ et du glucose extracellulaire. Notre modĂšle prĂ©dit quâen prĂ©sence dâune concentration saturante de glucose, la rĂ©orientation dans la membrane de SGLT1 suivant le relĂąchement intracellulaire de ses substrats est lâĂ©tape qui limite la vitesse de cotransport.The results presented in this thesis clarify certain functional aspects of the Na+/glucose cotransporter (SGLT1), a membrane protein which uses the downhill electrochemical gradient of Na+ ions to drive the accumulation of glucose in epithelial cells of the small intestine and the kidney.
We first used two microelectrodes electrophysiology on Xenopus oocytes to indentify the ionic species mediating the leak current of SGLT1, a current measured in the absence of glucose that is uncoupled from the strict 2 Na+/1 glucose stoichiometry
characterising cotransport. Our results showed that cations such as Li+, K+ and Cs+, which interact weakly with SGLT1 binding sites and are unable to generate the conformational changes that are triggered by Na+ binding, were however able to generate leak currents similar in amplitude to the one measured in the presence of Na+. This suggests that the leak current permeating through SGLT1 does so using a pathway that differs from the conformational changes associated with Na+/glucose cotransport. Moreover, it was found that the cationic leak and the passive water permeability could share a common pathway. We then sought to estimate the turnover rate of SGLT1 using the ion-trap technique, where a large tip ion-selective electrode (~100 ÎŒm) is pushed against the oocyte plasma membrane, thus enclosing a small volume of extracellular solution referred to as the trap. The variations in ionic concentration occurring in the trap as a consequence of SGLT1 activity made it possible to assess that the turnover rate of Na+/glucose cotransport was 13 s-1 when the membrane potential was clamped to -155 mV. As a last project, we focused our interest on the development of a kinetic model for SGLT1. Taking advantage of the simulated annealing algorithm, we constructed a 7-state kinetic scheme whose predictions accurately reproduced the currents of the cotransporter as a function of extracellular Na+ and glucose. According to our model, the rate limiting step of cotransport under a saturating glucose concentration is the reorientation of the empty carrier that follows the intracellular
release of substrates
Determination of the Na+/Glucose Cotransporter (SGLT1) Turnover Rate using the Ion-Trap Technique
Reply to âLetter to the editor: âThe use of extracellular, ion-selective microelectrodes to study the function of heterologously expressed transporters in Xenopus
The transport mechanism of the human sodium/myo-inositol transporter 2 (SMIT2/SGLT6), a member of the LeuT structural family
The Actual Ionic Nature of the Leak Current through the Na+/Glucose Cotransporter SGLT1
Expression of the Na+/glucose cotransporter SGLT1 in Xenopus oocytes is characterized by a phlorizin-sensitive leak current (in the absence of glucose) that was originally called a âNa+ leakâ and represents some 5â10% of the maximal Na+/glucose cotransport current. We analyzed the ionic nature of the leak current using a human SGLT1 mutant (C292A) displaying a threefold larger leak current while keeping a reversal potential (VR) of ââ15 mV as observed for wt SGLT1. VR showed only a modest negative shift when extracellular Na+ concentration ([Na+]o) was lowered and it was completely insensitive to changes in extracellular Clâ. When extracellular pH (pHo) was decreased from 7.5 to 6.5 and 5.5, VR shifted by +15 and +40 mV, respectively, indicating that protons may be the main charge carrier at low pHo but other ions must be involved at pHo 7.5. In the presence of 15 mM [Na+]o (pHo = 7.5), addition of 75 mM of either Na+, Li+, Cs+, or K+ generated similar increases in the leak current amplitude. This observation, which was confirmed with wt SGLT1, indicates a separate pathway for the leak current with respect to the cotransport current. This means that, contrary to previous beliefs, the leak current cannot be accounted for by the translocation of the Na-loaded and glucose-free cotransporter. Using chemical modification and different SGLT1 mutants, a relationship was found between the cationic leak current and the passive water permeability suggesting that water and cations may share a common pathway through the cotransporter