Study of mechanosensation using microbial TRP channels

描述(申请人提供)：检测触摸、听力、血压、器官伸展、渗透变化等机械刺激的分子仍知之甚少。另一方面，由于微生物为结晶学和分子操作提供的实验优势，对细菌的机械敏感通道MSCL进行了深入的分析。我们最近发现、克隆并表达了TrpY1，这是一种萌芽酵母的液泡膜通道，在膜片钳下是机械敏感的，在体内渗透向上移动时释放钙到细胞质中。TrpY1是Trp家族通道中的一员，其中一些通道与动物的机械感觉有关。我们将研究TrpY1是被第二信使激活，还是被邻近蛋白质的力激活，还是被脂双层的拉力激活。我们将测试纯化的TrpY1是否可以重组为脂双层并保持机械敏感性。TRPY1的真菌和古生菌同源物将被亚克隆、标记、表达和活性检测。我们将与Rees实验室合作，试图使这些纯TrpY蛋白质结晶，以解决它们的结构问题。动物色氨酸通道受多种因素激活或调节。因此，我们将测试TrpY1是否也受到各种因素的调节，如信号脂质。我们计划使用TrpY1作为一个模型来测试全身麻醉药是否会改变其活性，并寻找它们的结合部位。我们还将寻找可能与TrpY1相互作用的伙伴蛋白，包括那些可能需要的力量传递。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寻找TrpY1分子中在机械感觉中重要的部分。

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.