Functional characterization of urate handling by hSLC2A9 (hGLUT9) splice variants in a heterologous expression system

  • Author / Creator
    Witkowska, Katarzyna
  • Multiple, independent Genome Wide Association Studies have uncovered a significant correlation between abnormal plasma urate levels and single nucleotide polymorphisms within the hSLC2A9 gene. The gene product was originally characterized as a high affinity D-glucose and D-fructose transporter, which belongs to a larger family of facilitative transmembrane hexose transporters, named GLUTs. The two splice variants, hSLC2A9a and hSLC2a9b, were overexpressed in Xenopus laevis oocytes. A combination of radioisotope flux studies and electrophysiological analysis were employed to functionally characterize the urate-handling of these proteins. The two human hSLC2A9 (hGLUT9) isoforms mediate high capacity urate transport, which is selectively sensitive to benzbromarone. They display kinetic symmetry in their affinity and capacity to handle extracellular and intracellular urate. Surprisingly, urate uptake mediated by either isoform is not competitively inhibited by extracellular D-glucose and D-fructose over a wide range of concentrations. However, the transporters can exchange hexoses for urate, when the two substrates are placed on opposite sides of the membrane, as evidenced by trans-stimulation. Moreover, the two isoforms display different patterns of urate-transport modulation in response to trans-hexoses and kinase activators, indicating that functional differences between the two isoforms exist. Given that uric acid is approximately 90% dissociated under physiological pH, and exists as an organic anion (urate) in blood plasma, we investigated hSLC2A9’s capacity to carry current. Indeed, hSLC2A9a- and hSLC2A9b-expressing oocytes produce positive outward current in the presence of extracellular urate. This current does not appear to be sensitive to Na+, and is only moderately affected by Cl- depletion. Hence, we propose that the negatively charged urate is the only species contributing to the electrogenicity of hSLC2A9. Given that the membrane potential is negative inside cells, we propose that both variants mediate urate efflux under physiological conditions. Both isoforms of hSLC2A9 are expressed in opposite membrane domains of human proximal tubule epithelium. We propose a model for renal handling of urate in humans which explains how hypouricemia and hyperuricemia can be associated with the same gene product, and which may provide new treatment opportunities of gout, hypertension and metabolic syndrome.

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  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
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  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Physiology
  • Supervisor / co-supervisor and their department(s)
    • Cheeseman, Chris (Physiology)
  • Examining committee members and their departments
    • Carruthers, Anthony (Biochemistry and Molecular Pharmacology, MassU)
    • Goss, Greg (Biological Sciences)
    • Young, James (Physiology)
    • Alexander, Todd (Physiology, Paediatrics)
    • Chen, Xing-Zhen (Physiology)