Study of mechanosensation using microbial TRP channels

利用微生物 TRP 通道进行机械感觉研究

• 批准号: 6871631
• 负责人: CHING KUNG
• 金额: $ 30.12万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6871631
• 关键词: Escherichia coli; bacterial genetics; bacterial proteins; cell osmotic pressure; crystallization; genetic techniques; lipid bilayer membrane; mechanical pressure; membrane permeability; membrane proteins; molecular cloning; mutant; protein protein interaction; protein structure function; second messengers

DESCRIPTION (provided by applicant): The molecules that detect the mechanical stimuli for touch, hearing, blood pressure, organ extension, osmotic changes etc. are still poorly understood. On the other hand; the bacterial mechanosensitive channel MscL has been analyzed in depth because of the experimental advantages microbes offer for crystallography and molecular manipulations. We have recently discovered, cloned and expressed TrpY1, a vacuolar-membrane channel of the budding yeast that is mechanosensitive under patch clamp, and releases Ca 2+ into the cytoplasm upon osmotic up-shift in vivo. TrpY1 is a member of the TRP family channels, some of which have been associated with mechanosensations in animals. We will examine whether TrpY1 is activated by second messengers, by force from neighboring proteins, or by stretch force from the lipid bilayer. We will test whether purified TrpY 1 can be reconstituted into lipid bilayers and remain mechanosensitive. Fungal and archaeal homologs of TRPY1 will be subcloned, tagged, expressed, and examined for activities. We will attempt to crystallize these pure TrpY proteins towards solving their structures in collaboration with the Rees laboratory. Animal TRP channels are each activated or regulated by multiple factors. We will therefore test whether TrpY1 is also regulated by various factors such as signaling lipids. We plan to use TrpY1 as a model to test whether general anesthetics may change its activities and to find their binding site. We will also look for possible partner proteins that interact with TrpY1, including those that may be needed for force transmission. Taking advantage of the facile and powerful genetic manipulations, we plan to use forward and reverse genetics to find the parts of the TrpY1 molecule that are important in mechanosensation.

描述（由申请人提供）：检测触觉、听觉、血压、器官伸展、渗透变化等机械刺激的分子仍然知之甚少。另一方面，由于微生物为晶体学和分子操作提供的实验优势，细菌机械敏感通道MscL已被深入分析。我们最近发现，克隆和表达的TrpY 1，一个液泡膜通道的芽殖酵母，是机械膜片钳下敏感，并释放Ca2+进入细胞质时，渗透上移体内。TrpY1是TRP家族通道的成员，其中一些与动物的机械感觉有关。我们将研究TrpY1是否被第二信使激活，通过邻近蛋白质的力，或通过脂质双层的拉伸力。我们将测试纯化的TrpY 1是否可以重组成脂质双层并保持机械敏感性。TRPY1的真菌和古细菌同源物将被亚克隆、标记、表达并检查活性。我们将尝试与里斯实验室合作，将这些纯TrpY蛋白结晶化，以解决其结构问题。动物TRP通道各自被多种因素激活或调节。因此，我们将测试TrpY1是否也受到信号脂质等各种因素的调节。我们计划使用TrpY 1作为模型，以测试是否全身麻醉药可能会改变其活动，并找到它们的结合位点。我们还将寻找与TrpY1相互作用的可能的伴侣蛋白，包括那些可能需要力传递的蛋白。利用简单而强大的遗传操作，我们计划使用正向和反向遗传学来找到TrpY1分子中对机械感觉重要的部分。