| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
In skeletal muscle, voltage-gated calcium channel (dihyrdopyridine receptor or DHPR) and ryanodine receptor (RyR) play critical roles for excitation-contraction coupling.The voltage-gated calcium channel is a sensor molecule of the membrane voltage and controls the opening of the ryanodine receptor.Although these two molecules are thought to interact directly, there is little evidence of the physical protein-protein interaction.The aim of this project is to study the protein-protein interaction between the DHPR and the RyR.For this purpose, we used the yeast two-hybrid system.
Project 1 : Test for the interaction between DHPR and RyR.
A small piece of the DHPRalpha1s subunit (amino acid number ; 719-768) was inserted into the DNA-binding (DB) plasmid to yield s53.Likewise, a piece of the type-1 ryanodine receptor (RyR-1) and the type-2 ryanodine receptor (RyR-2) were inserted into the activation-domain (AD) plasmid to yield sRl6(RyR-1 ; 1837-2168) and cRl6(RyR-2 ; 1817-2142), respectivel
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y.These plasmids were co-transfected and His^+ cells were selected.Results showed that only the yeast co-transfected with s53 and sRl6 could grow in synthetic minimum medium.However, this combination did not show positive beta-galactosidase activity.Because of the discrepancy between the nutrition-requirement test and the beta-galactosidase-activity test, we could not get clear evidence of the physical interaction between the DHPR and the RyR.
Project 2 : Screening of new proteins which may bind to the DHPR.
To screen new proteins which may bind to the DHPR, we constructed a cDNA library from the skeletal muscle which was inserted into the AD plasmid and tested interaction with five DB plasmids which include internal loops of the DHPR.First, using the I-II loop of the DHPR, we isolated the beta subunit of the DHPR.This is consistent with previous results and indicates that the screening system works well.Next, we found that the troponin T binds to the C-terminal potion of the DHPR.The function of the troponin T to the DHPR is not clear so far.However, there is the possibility of the troponin T to localize the ion channels. Less
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