December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
Water Permeability of Pathogenic Mutants of Human Facilitative Glucose Transporter Glut1
Author Affiliations & Notes
  • L Ma
    Ophthalmology
    Columbia Univ New York NY
  • D Wang
    Neurol
    Columbia Univ New York NY
  • FA Zuniga
    Ophthalmology
    Columbia Univ New York NY
  • K Kuang
    Ophthalmology
    Columbia Univ New York NY
  • H Yang
    Neurol
    Columbia Univ New York NY
  • P Iserovich
    Ophthalmology
    Columbia Univ New York NY
  • JM Pascual
    Neurol
    Columbia Univ New York NY
  • DC DeVivo
    Neurol
    Columbia Univ New York NY
  • J Fischbarg
    Ophthalmology
    Columbia Univ New York NY
  • Footnotes
    Commercial Relationships   L. Ma, None; D. Wang, None; F.A. Zuniga, None; K. Kuang, None; H. Yang, None; P. Iserovich, None; J.M. Pascual, None; D.C. DeVivo, None; J. Fischbarg, None. Grant Identification: Support: EY08918(JF), NS37949 and NS01698(DCD), RPB, and Fight for Sight (LM)
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 3257. doi:
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      L Ma, D Wang, FA Zuniga, K Kuang, H Yang, P Iserovich, JM Pascual, DC DeVivo, J Fischbarg; Water Permeability of Pathogenic Mutants of Human Facilitative Glucose Transporter Glut1 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3257.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: We have reported a 3D model of the human facilitative glucose transporter GLUT1 (JBC:2001;276:44970-5) that contains two channels, a main one for glucose transport, and an auxiliary one possibly for the transport of water and other substrates. We have previously described that the pathogenic Glut1 mutant T310I which underlies the glucose transporter deficiency syndrome (Glut1 DS) showed lack of glucose transport and increased osmotic water permeability (Pf ). Here we correlate the locations of other pathogenic mutants with their transport characteristics by expression of mutants and wild-type Glut1 cRNA in Xenopus oocytes. Methods: After in vitro transcription, Xenopus laevis oocytes were micro-injected with 50 nl of water or 50 nl (50 ng) of cRNA encoding wild-type or 6 missense mutants of Glut1 DS (S66F, R126H, E146K, K256V, T310I and R333W). Three days after micro-injection, glucose transporter activity was determined by 2-deoxy-D-[3H]glucose (deoxyglucose, DOG) uptake by the oocytes. Pf was determined from the rate of volume increase on exposure to hyposmotic medium (178 to 15 mOsm). Transient oocyte volume changes were monitored automatically using an image analysis system (resolution: 6s). Results: DOG (concentration: 2 mM) uptake (nmol/10 min/oocyte) was: wild-type, 1.31 0.05; S66F, 0.06 0.004; R126H, 0.08 0.007; E146K, 0.49 0.02; K256V, 0.06 0.003; T310I, 0.11 0.007; R333W, 0.16 0.01; and H2O, 0.01 0.001, respectively. On the other hand, The corresponding Pf values (in µm/s) were: wild-type, 25.58 0.70; S66F, 37.6 1.76; R126H, 12.1 0.60; E146K, 13.19 0.34; K256V, 14.72 0.32; T310I, 37.5 1.9; R333W, 20.66 0.74; and H2O, 13.24 0.74, respectively. Conclusion: All of the pathogenic mutants show lack of glucose transport, but only pathogenic mutants S66F and T310I have high Pf. According to our model, T310 is located on the main channel, and its replacement by I may obstruct the channel, which doesn't allow glucose to pass and would immobilize the auxiliary channel open for water transport. S66 is instead on the auxiliary channel; replacement by phenylalanine could open the auxiliary channel for water but not allow the conformation changes necessary for glucose transport. Most interestingly, the other three residues (256, 272, and 333) are in intracellular loops, and configure a triangle (10.19 x 10.01 x 13.77 Å between Cα atoms), so they could possibly bind an indispensable factor. CR: N Support: EY08918(JF), NS37949 and NS01698(DCD), RPB, and Fight for Sight (LM)

Keywords: 446 ion transporters • 467 metabolism • 527 protein structure/function 
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