December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
Interaction of cGMP-gated Channel with Peripherin-2 and Its Role in Linking Discs to the Plasma Membrane in Photoreceptor Rod Outer Segments
Author Affiliations & Notes
  • LL Molday
    Department of Biochemistry and Molecular Biology University of British Columbia Vancouver BC Canada
  • A Poestch
    Department of Biochemistry and Molecular Biology University of British Columbia Vancouver BC Canada
  • RS Molday
    Department of Biochemistry and Molecular Biology University of British Columbia Vancouver BC Canada
  • Footnotes
    Commercial Relationships   L.L. Molday, None; A. Poestch, None; R.S. Molday, None. Grant Identification: NEI Grant EY02422
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 1399. doi:
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      LL Molday, A Poestch, RS Molday; Interaction of cGMP-gated Channel with Peripherin-2 and Its Role in Linking Discs to the Plasma Membrane in Photoreceptor Rod Outer Segments . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1399.

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

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Abstract

Abstract: : . Purpose: The rod outer segment (ROS) consists of a plasma membrane (PM) that encloses a highly ordered stack of hundreds of discs. Protein-protein interactions are known to maintain the defined spacing between the discs and PM and between individual discs. However, the identity of the proteins involved in these membrane associations is not known. The goal of this study was to determine if the GARP part of the cGMP-gated channel interacts with the peripherin-2:rom-1 complex or other disc specific proteins to maintain and stabilize the unique ROS structure. Methods: Membrane proteins were solubilized from untreated or crosslinked ROS or from transiently transfected HEK-293 cells. Highly specific monoclonal antibodies were used with immunoprecipitation and Western blotting techniques to identify interacting proteins. Immunocytochemical techniques were employed to localize proteins within rod photoreceptors and HEK-293 cells. Results: When untreated or crosslinked ROS membranes were subjected to immunoprecipitation on a channel-immunoaffinity matrix, peripherin-2, rom-1 and the Na/Ca-K exchanger were found to co-precipitate with the α and ß-subunits of the cGMP-gated channel. In contrast, ABCR and guanylate cyclase (Ret GC1) did not co-precipitate with the channel. GARP-1 and GARP-2 proteins also associated with peripherin-2:rom-1 oligomers, but not with the cGMP-gated channel. When ROS were treated with trypsin under conditions known to separate the discs from the PM, the GARP part of the channel ß-subunit was degraded. The cGMP-gated channel and the peripherin-2 complex has been expressed in HEK-293 cells. This cell system is currently being used to further evaluate peripherin-2-channel interactions. Conclusion: The cGMP-gated channel associates with the Na/Ca-K exchanger to form a complex in the PM of ROS. This complex interacts with peripherin-2:rom-1 oligomers present in the rim region of disc membranes. These studies suggest a model in which the GARP part of the cGMP-gated channel associates with peripherin-2-rom-1 oligomers within the narrow cytoplasmic space between the disc and PM. This interaction is thought to contribute to the formation and stabilization of the ROS and to the anchoring of the channel:exchanger complex in the PM.

Keywords: 517 photoreceptors • 526 protein purification and characterization • 445 ion channels 
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