Protein-protein interaction between voltage-gateal calcium channel and ryanodine recegtor.

在骨骼肌中，电压门控钙通道（二氢吡啶受体或DHPR）和ryanodine受体（RyR）在兴奋-收缩耦合中起关键作用。电压门控钙通道是膜电压的传感器分子，控制着ryanodine受体的打开。虽然这两种分子被认为是直接相互作用的，但很少有证据表明蛋白质与蛋白质之间存在物理相互作用。本项目旨在研究DHPR与RyR之间的蛋白-蛋白相互作用。为此，我们使用酵母双杂交系统。项目1：检测DHPR与RyR的相互作用。将一小段DHPRalpha1s亚基（氨基酸编号719-768）插入dna结合（DB）质粒中，得到s53。同样，将1型ryanodine受体（RyR-1）和2型ryanodine受体（RyR-2）片段插入到激活域（AD）质粒中，分别得到sr16 （RyR-1; 1837-2168）和cr16 （RyR-2; 1817-2142）。结果表明，只有s53和sr16共转染的酵母才能在合成最小培养基中生长。然而，这种组合没有显示出阳性的-半乳糖苷酶活性。由于营养需求试验与β -半乳糖苷酶活性试验的差异，我们无法得到DHPR与RyR之间物理相互作用的明确证据。项目2：筛选可能与DHPR结合的新蛋白。为了筛选可能与DHPR结合的新蛋白，我们从骨骼肌中构建了cDNA文库，并将其插入到AD质粒中，并测试了与包含DHPR内环的5个DB质粒的相互作用。首先，利用DHPR的I-II环，我们分离了DHPR的β亚基。这与先前的结果一致，表明筛选系统工作良好。接下来，我们发现肌钙蛋白T与DHPR的c端药水结合。肌钙蛋白T对DHPR的作用目前还不清楚。然而，肌钙蛋白T有可能定位离子通道。少

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 … More 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