Publication Date

2012-10-01

Availability

Open access

Embargo Period

2012-10-01

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Physiology and Biophysics (Medicine)

Date of Defense

2012-09-14

First Committee Member

Ana Diez-Sampedro

Second Committee Member

Peter Larsson

Third Committee Member

Gerhard Dahl

Fourth Committee Member

Charles Luetje

Fifth Committee Member

Christof Grewer

Abstract

There are two main types of sugar transporters: SGLTs which are Na+-dependent glucose transporters and GLUTs which are facilitative glucose transporters. SGLT1 is the most widely studied SGLT protein. It is a glucose/galactose transporter highly expressed in the small intestine and associated with glucose-galactose malabsorption disease (GGM). SGLT2 is a glucose transporter highly expressed in the kidney and associated with familial renal glucosuria (FRG). There is a growing interest in development of human SGLT2 inhibitors to treat patients with diabetes by lowering blood glucose levels. Human SGLT3 (hSGLT3), on the other hand, is not as well characterized as SGLT1 or SGLT2. Unlike other SGLT proteins, hSGLT3 is a glucose sensor and not a glucose transporter. While only one gene codes for SGLT3 in humans, two genes in mouse code for two SGLT3 proteins, type 3a (mSGLT3a) and type 3b (mSGLT3b). Our lab cloned mSGLT3b from kidney and recently characterized its activity. However, information on mSGLT3a is lacking. The aims of this study are two-fold: (1) to functionally characterize mSGLT3a and (2) to determine the role of residue 457 in the function of mSGLT3a. Results for Aim 1 are presented in Chapter 2. Results in Aim 2 are presented in Chapter 3. We have cloned mSGLT3a from small intestine. We characterized its function by expressing mSGLT3a in Xenopus laevis oocytes and performing electrophysiology and sugar transport assays. We found that, like hSGLT3, mSGLT3a does not transport sugar. We also found that sugars do not induce currents at pH 7.4, but induce currents at pH 5. Currents observed in acidic pH increases with increasing extracellular proton concentrations. Expression and characterization of mSGLT3a in mammalian cells confirmed our findings with an oocyte expression system. In addition, we have cloned rSGLT3a from small intestine and found that, like mSGLT3a, acidic pH results in currents that increase in the presence of glucose. We mutated residue 457 from glutamate (E) to glutamine (Q), E457Q-mSGLT3a, to recapitulate the residue at this position in SGLT1 proteins. E457Q-mSGTL3a exhibited a Na+-dependent transport of sugar that was inhibited by phlorizin. Residue 457 has an important functional role in SGLT proteins. In hSGLT1, a mutation in this residue in from glutamine (Q) to arginine (R), Q457R, causes GGM. The crystal structure of vibrio SGLT showed that the corresponding residue directly binds the sugar substrate. Although E457Q-mSGLT3a transported sugar, glucose-induced currents at pH 7.4 were not observed. Residue 457 in SGLT proteins has been associated with changes in Na+ binding, sugar binding and transport, and charge movement across the protein. We explored the role of residue 457 in ion permeability in both E457Q-mSGLT3a and WT-mSGLT3a. We analyzed reversal potentials of proton-activated currents at various conditions. Our results suggest that WT-mSGLT3a is a proton-gated ion channel permeable to K+, Na+, H+, and Cl- ions. We also showed that WT-mSGLT3a is selective for H+. In E457Q-mSGLT3a, neutralization of a negatively charged amino acid at position 457 resulted in an increase in Cl- conductance and a loss of permeability to H+ ions.

Keywords

acid sensor; pH; sodium-glucose co-transporter; SGLT3

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