The goal of this project is to understand the mechanisms of intestinal sugar absorption. The major intestinal sugar transport proteins (SGLT1, GLUT5, and GLUT2) have been cloned sequenced and expressed, and the genes have been mapped and sequenced. Here we propose to examine the mechanism of sugar transport by expressing cloned Na/glucose cotransporters in a simple model system, Xenopus laevis oocytes, and measuring the bioenergetics and kinetics of transport. The experiments will be designed to critically evaluate our quantitative model for Na/glucose cotransport, and the results will be used to refine, extend, or reject the model. Electrophysiological and radioactive tracer techniques will be used to measure study-state kinetic parameters, and new voltage-jump and concentration-jump techniques will be used to measure pre-steady-state kinetics in the milli- and micro- second time scales. Charge transfer measurements have enabled us to predict the number of cotransporters expressed in the oocyte plasma membrane, up to 40,000/um2, and this assay will be used to obtain structural information about the transport protein. Similar kinetic and structural studies will be carried out on mutant proteins, e.g. the 15 missense mutants of SGLT1 that cause glucose-galactose malabsorption in patients.
The second aim will be achieved by measuring the kinetic properties of cloned sugar transporters SGLT1 (human, rabbit, rat, pig, mouse and sheep), SGLT2, and SMIT (Na/myoinositol), expressed in oocytes, correlating the structure of the transporters with their kinetic properties, and making site-directed mutations and chimeras to test our conclusions.
the third aim i s to determine the role of a low affinity Na/glucose cotransporter, SGLT2, in intestinal sugar transport. The distribution and function of SGLT2 in the intestine, kidney, liver, spleen, and skeletal muscle will be determined using Western Blots, Northern Blots, immunocytochemistry and transport assays on isolated cells and membranes. Overall, our studies should provide new insights into the biophysics of transport, and the mechanisms of intestinal sugar absorption.
| Kepe, Vladimir; Scafoglio, Claudio; Liu, Jie et al. (2018) Positron emission tomography of sodium glucose cotransport activity in high grade astrocytomas. J Neurooncol 138:557-569 |
| Paz, Aviv; Claxton, Derek P; Kumar, Jay Prakash et al. (2018) Conformational transitions of the sodium-dependent sugar transporter, vSGLT. Proc Natl Acad Sci U S A 115:E2742-E2751 |
| Gorraitz, Edurne; Hirayama, Bruce A; Paz, Aviv et al. (2017) Active site voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1. Proc Natl Acad Sci U S A 114:E9980-E9988 |
| Ghezzi, Chiara; Yu, Amy S; Hirayama, Bruce A et al. (2017) Dapagliflozin Binds Specifically to Sodium-Glucose Cotransporter 2 in the Proximal Renal Tubule. J Am Soc Nephrol 28:802-810 |
| Sala-Rabanal, Monica; Hirayama, Bruce A; Ghezzi, Chiara et al. (2016) Revisiting the physiological roles of SGLTs and GLUTs using positron emission tomography in mice. J Physiol 594:4425-38 |
| Zeuthen, Thomas; Gorraitz, Edurne; Her, Ka et al. (2016) Structural and functional significance of water permeation through cotransporters. Proc Natl Acad Sci U S A 113:E6887-E6894 |
| Adelman, Joshua L; Ghezzi, Chiara; Bisignano, Paola et al. (2016) Stochastic steps in secondary active sugar transport. Proc Natl Acad Sci U S A 113:E3960-6 |
| Gallo, Linda A; Wright, Ernest M; Vallon, Volker (2015) Probing SGLT2 as a therapeutic target for diabetes: basic physiology and consequences. Diab Vasc Dis Res 12:78-89 |
| Scafoglio, Claudio; Hirayama, Bruce A; Kepe, Vladimir et al. (2015) Functional expression of sodium-glucose transporters in cancer. Proc Natl Acad Sci U S A 112:E4111-9 |
| Adelman, Joshua L; Sheng, Ying; Choe, Seungho et al. (2014) Structural determinants of water permeation through the sodium-galactose transporter vSGLT. Biophys J 106:1280-9 |
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