1988 Fiscal Year Final Research Report Summary
Reconstitution of phototransduction channel from frog rod outer segments in planar lipid bilayers
| Project/Area Number |
62570041
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General physiology
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| Research Institution | Nagoya University School of Medicine |
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Principal Investigator |
SOKABE Masahiro Nagoya University School of Medicine, Associate Prof., 医学部, 助教授 (10093428)
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| Co-Investigator(Kenkyū-buntansha) |
SHINOZAWA Takao Faculty of Engineering,Gunma University,Associate Prof., 工学部, 助教授 (30025449)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Frog / Photoreceptor / Cyclic GMP / Ion channel / Photoaffinity labeling / planar bilayer / Purification |
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| Research Abstract |
Vertebrate photoreceptors show hyperporalizing responses to light stimulations due to the closure of cGMP-dependent cation channels on outer segment membranes. The aim of this project is to identifiy and purify the cGMP-dependent ion channel protein. Integral membrane protein fraction from frog rod outer segments was labeled with [^3H]cGMP and four cGMP binding proteins with molecular weights of 250K, 100K, 92K, and 66K were identified. The incoropration of the fraction into planar lipid bilayers resulted in the appearance of at least three kinds of channel activity; two of them were related to cGMP. Since the 100K and 92K were phosphodiesterase, the 250K and/ar 66K proteins were strongly suggested to be cGMP dependent channel proteins. We performed partial purification of the 250K protein and succeeded in reconstituting of it into liposomes. By using polyclonal antibody (IGG) against the 250K protein the distribution of the protein was investagted by means of immuno-electronmicroscopy. The 250K protein seemed to be enriched on disc membranes rather than plasma membranes, which may be in harmony with recent reports indicating an enrichment of cGMP-dependent channels in disc membranes. Interestingly the IgG fraction reacts with both 250K and 66K proteins suggesting that the former is a precurser of the latter. It may also be possible these proteins are different types of cGMP-dependent channels. These problems would be solved by the further purification and reconstitution of these proteins.
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