The long term objective of this research program is to study the molecular structure and properties of rod outer segment (ROS) disk and plasma membrane proteins in order to elucidate their role in visual excitation, in the organization and renewal of the ROS organelle and in retinal degenerative diseases. Four related areas of research will be investigated in this grant period: 1) The ROS plasma membrane and plasma membrane specific proteins will be purified and characterized. This will be carried out by labeling ROS plasma membrane specific glycoproteins with recently developed Ricin-gold-dextran particles and separating the 'dense' labeled plasma membrane from disks by gradient centrifugation. The protein and lipid composition of the plasma membrane will be determined and compared with disk membranes using biochemical, immunochemical and electron microscopic techniques developed over the past grant period. Emphasis will be directed toward identifying and characterizing the c-GMP binding protein, Na+ channels and ROS plasma membrane-specific glycoproteins. The generation of monoclonal antibodies (Mab) against these proteins for use as specific probes for studying their distribution, structure and function will be carried out. 2) ROS spectrin recently identified in this laboratory will be purified by gel filtration, ion exchange and immunoaffinity chromatography and characterized with regard to its subunit structure by SDS-gel electrophoresis, its size and shape by Platinum rotary shadowing for electron microscopy and its interaction with other membrane and cytoskeletal proteins by binding analysis and reconstitution methods. The possible role of ROS spectrin and other cytoskeletal proteins in disk-disk and disk-plasma membrane interactions will also be studied. (3)Peripherin, a newly discovered ROS rim protein, will be further characterized using biochemical and DNA recombinant techniques. The gene will be cloned and its nucleotide and amino acid sequence determined in order to obtain information about its membrane organization and relation to other known proteins. 4) Antirhodopsin Mabs will be further characterized and used as immunological probes to analyze the structural and functional domains of rhodopsin.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY002422-10
Application #
3256755
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1978-04-01
Project End
1990-05-31
Budget Start
1987-06-08
Budget End
1988-05-31
Support Year
10
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of British Columbia
Department
Type
DUNS #
800772162
City
Vancouver
State
BC
Country
Canada
Zip Code
V6 1Z3
McMillan, Hugh J; Telegrafi, Aida; Singleton, Amanda et al. (2018) Recessive mutations in ATP8A2 cause severe hypotonia, cognitive impairment, hyperkinetic movement disorders and progressive optic atrophy. Orphanet J Rare Dis 13:86
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Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J et al. (2016) PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina. Mol Ther Methods Clin Dev 3:16051
Vinberg, Frans; Wang, Tian; Molday, Robert S et al. (2015) A new mouse model for stationary night blindness with mutant Slc24a1 explains the pathophysiology of the associated human disease. Hum Mol Genet 24:5915-29

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