May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Structure of TRPV2 by Cryo-Electron Microscopy
Author Affiliations & Notes
  • V. Y. Moiseenkova-Bell
    Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas
  • I. Serysheva
    Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas
  • T. G. Wensel
    Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas
  • Footnotes
    Commercial Relationships V.Y. Moiseenkova-Bell, None; I. Serysheva, None; T.G. Wensel, None.
  • Footnotes
    Support R01-EY07981; T90-DK070109 HIGHWIRE EXLINK_ID="48:5:2963:1" VALUE="DK070109" TYPEGUESS="GEN" /HIGHWIRE ; Welch Foundation Q-0035; National Center for Macromolecular Imaging P41-RR002250 HIGHWIRE EXLINK_ID="48:5:2963:2" VALUE="RR002250" TYPEGUESS="GEN" /HIGHWIRE
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2963. doi:
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    • Get Citation

      V. Y. Moiseenkova-Bell, I. Serysheva, T. G. Wensel; Structure of TRPV2 by Cryo-Electron Microscopy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2963.

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

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Abstract

Purpose:: TRPV2 is a cation channel expressed in the mammalian retina. It is relatively non-selective, with fairly high calcium permeability, and is reported to be regulated by diacyl glycerol. Our goal is to determine the three-dimensional structures of TRP family channels, including TRPV2, in order to understand the relationships between structure and function, and as an aid in the development of TRP channel-directed therapies.

Methods:: Human TRPV2 was expressed in the budding yeast, Saccharomyces cerevisiae, by transient transfection and immuno-affinity purified in detergent using an epitope tag engineered into its carboxyl terminus. Cryo-electron microscopy and single-particle analysis using the software suites EMAN and IMAGIC were used to generate three-dimensional reconstructions of the channel protein with and without a monoclonal antibody Fab bound to its carboxyl terminus. The CHIMERA package was used for manual fitting of x-ray structures of structurally related potassium channels.

Results:: As revealed by cryo-electron microscopy, TRPV2 is a tetrameric protein with two large domains connected together in a "hanging gondola" motif similar to that observed for other cation channels, including TRPV1 and TRPY1. The cytoplasmic carboxyl terminus is located near the base of one of the large domains.

Conclusions:: Expression in yeast and immuno-affinity purification is a powerful approach to obtaining large quantities of TRPV2 and other TRP channels for structural and functional studies. TRP channels have a common architecture resembling that of potassium channels in the transmembrane segments.

Keywords: ion channels • protein structure/function • microscopy: electron microscopy 
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