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

电压门钙通道和兰尼碱受体之间的蛋白质-蛋白质相互作用。

• 批准号: 09670064
• 负责人: NAKAI Junichi
• 金额: $ 1.98万
• 依托单位: Okazaki National Research Institutes
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670064/
• 关键词: ryanodine receptor; calcium release Channel; voltage-gated calcium channel; molecular biology; dihydropyridine receptor; excitation-contraction coupling

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

在骨骼肌中，电压门控钙通道（二氢吡啶受体或DHPR）和兰尼碱受体（RyR）在兴奋-收缩耦合中发挥关键作用。电压门控钙通道是膜电压的传感器分子，控制兰尼碱受体的开放。虽然这两个分子被认为直接相互作用，但很少有证据表明 该项目的目的是研究 DHPR 和 RyR 之间的蛋白质 - 蛋白质相互作用。为此，我们使用了酵母双杂交系统。项目 1：测试 DHPR 和 RyR 之间的相互作用。将一小段 DHPRalpha1s 亚基（氨基酸编号；719-768）插入到 DNA 结合（DB）质粒中 产生 s53。同样，将 1 型兰尼碱受体 (RyR-1) 和 2 型兰尼碱受体 (RyR-2) 插入激活域 (AD) 质粒，分别产生 sRl6(RyR-1 ; 1837-2168) 和 cRl6(RyR-2 ; 1817-2142) ... 更多y.这些质粒被共转染并 选择His^+细胞。结果显示，只有s53和sRl6共转染的酵母才能在合成基本培养基中生长。然而，这种组合没有表现出阳性的β-半乳糖苷酶活性。由于营养需求测试和β-半乳糖苷酶活性测试之间的差异，我们无法获得DHPR与β-半乳糖苷酶活性测试之间的物理相互作用的明确证据。 RyR.项目2：筛选可能与DHPR结合的新蛋白质。为了筛选可能与DHPR结合的新蛋白质，我们从骨骼肌构建了一个cDNA文库，将其插入AD质粒中，并测试了与包含DHPR内部环的五个DB质粒的相互作用。首先，使用DHPR的I-II环，我们分离了DHPR的β亚基 DHPR。这与之前的结果一致，表明筛选系统运行良好。接下来，我们发现肌钙蛋白T与DHPR的C端部分结合。肌钙蛋白T对DHPR的功能目前尚不清楚。但是，肌钙蛋白T有可能定位离子通道。较少的

项目成果

Robert T.Dirksen: "The S5-S6 linker of repeatl is a critical determinant of L-type Ca^<2+> channel conductance." J.Biophys.73. 1402-1409 (1997)

Robert T.Dirksen：“repeat1 的 S5-S6 连接子是 L 型 Ca^<2> 通道电导的关键决定因素。”

Jusus Gavcia: "Role of S4 Segments and the Leucine Heptad Motif in the Activation of an L-type Calcium Channel" J.Biophys.72. 2515-2523 (1997)

Jusus Gavcia：“S4 片段和亮氨酸七肽基序在 L 型钙通道激活中的作用”J.Biophys.72。

Junichi Nakai: "Two regions of the ryanodine receptor involved in coupling with L-type Ca^<2+> channels." J.Boil.Chem.273. 13403-13406 (1998)

Junichi Nakai：“兰尼碱受体的两个区域参与与 L 型 Ca^2 通道的偶联。”

Yasuyo Nakashima: "Molecular cloning and characterization of a human brain ryanodine receptor" FEBS Lett.417. 157-162 (1997)

Yasuyo Nakashima：“人脑兰尼碱受体的分子克隆和表征”FEBS Lett.417。

Junichi Nakai: "Localization in the II-III loop of the dihydropyridine receptor of a sequence critical for excitation-contraction coupling" J.Biol.Chem.273. 24983-24986 (1998)

Junichi Nakai：“二氢吡啶受体 II-III 环中对兴奋-收缩耦合至关重要的序列的定位”J.Biol.Chem.273。