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A novel signal transduction mechanism via intracellular ATP sensor

通过细胞内 ATP 传感器的新型信号转导机制

基本信息

批准号：
09660086
负责人：
UEDA Kazumitsu
金额：
$ 0.7万
依托单位：
KYOTO UNIVERSITY
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-09660086/
关键词：
ABC proteins transporter K^+ channel CFTR MDRI SUR signal transductron ATP P-糖タンパク質 MDR MRP 糖尿病癌 ABCス-パファミリー k^+チャネル多剤耐性分子生物学生化学

项目摘要

ATP-binding cassette (ABC) proteins comprise a family of structurally related membrane proteins sharing well conserved nucleotide binding domains. They include MDR1, an ATP-dependent efflux pump that extrude toxic xenobiotics, the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel, and the sulfonylurea receptor (SUR 1), the pancreatic ATP-sensitive K^+ channel regulator.The objective of the research was1) to develop a deep understanding of the molecular mechanisms of transporters, channels, and regulators of ABC proteins.2) to understand the molecular mechanisms of having dual functions.3) to explore the physiological functions of ABC proteins in cellular signal transduction mechanism.The K_<ATP> channels in pancreatic beta-cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of K_<ATP> channels by ATP, MgADP, and the pharmacological agents, the molecular mechanism … More of K_<ATP> channel regulation remains unclear. The K_<ATP> channel is a hetero-oligomeric complex of SUR 1 subunits of ABC superfamily with two nucleotide- binding folds and the pore-forming Kir6.2 subunits. We revealed that MgATP and MgADP, but not MgATP-gammaS, stabilize binding of prebound 8-azido-[alpha-^<32>P]ATP to SUIR1. Mutation in the Walker A and B motifs of NBF2 of SUIR1 abolished this stabilizing effect of MgADP.These results suggest that SURl binds 8-azido-ATP strongly at NBF1, and that MgADP, either by direct binding to NBF2 or hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP binding at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azido-[alpha-^<32>P]ATP from SUR1 in the presence of MgADP or MgATP in a concentration-dependent manner. This direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP, and, in cooperation with the MgADP bound at NBF2, causes ATP to be released from NBF1. Less

三磷酸腺苷结合盒(ATP-bindingcassette，ABC)蛋白是一类结构相关的膜蛋白，具有保守的核苷酸结合域。它们包括分泌有毒外源物质的依赖于ATP的外排泵MDR1、阴离子通道囊性纤维化跨膜电导调节器(CFTR)和胰腺ATP敏感的K~(++)通道调节剂磺脲受体(SUR_1)。研究的目的是：1)深入了解转运体、通道和ABC蛋白调节器的分子机制；2)了解ABC蛋白具有双重功能的分子机制；3)探讨ABC蛋白在细胞信号转导机制中的生理功能。胰腺β细胞中的K_和lt；ATP和GT；通道在葡萄糖诱导的胰岛素分泌调节中起关键作用。虽然电生理学研究为三磷酸腺苷、二磷酸镁和药物对钾通道的复杂控制提供了线索，但分子机制…K&lt；ATP；通道监管的更多内容仍不清楚。K_&lt；ATP&gt；通道是ABC超家族中具有两个核苷酸结合折叠的SuR_1亚基和形成孔道的Kir6.2亚基的异源低聚复合体。我们发现，镁ATP和镁ADP，而不是镁ATP-伽马，稳定了预结合的8-叠氮基-[α-^&lt；32&gt；P]ATP与SuIR1的结合。SuIR1的NBF2的Walker A和B基序的突变取消了镁的这种稳定作用。这些结果表明，Surl在NBF1处与8-叠氮基-ATP强烈结合，而镁ADP通过直接与NBF2结合或在NBF2处水解结合的镁ATP来稳定NBF1处的预结合的8-叠氮基-ATP结合。磺脲类药物格列本脲以浓度依赖的方式引起SUR1细胞8-叠氮基-[α-叠氮基]-[α-P]-[32&P]ATP的释放。这一直接的生化证据表明，SUR1的两个NBF在核苷酸结合中存在协同作用，这表明格列本脲既能阻断ATP和镁ADP的协同结合，又能与NBF2上的镁ADP结合，导致ATP从NBF1中释放。较少