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. ? ? ?
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