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. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047856-11
Application #
7148690
Study Section
Biophysics of Synapses, Channels, and Transporters Study Section (BSCT)
Program Officer
Shapiro, Bert I
Project Start
1994-05-01
Project End
2008-11-30
Budget Start
2006-12-01
Budget End
2007-11-30
Support Year
11
Fiscal Year
2007
Total Cost
$290,407
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Loukin, Stephen; Su, Zhenwei; Kung, Ching (2011) Increased basal activity is a key determinant in the severity of human skeletal dysplasia caused by TRPV4 mutations. PLoS One 6:e19533
Loukin, Stephen; Zhou, Xinliang; Su, Zhenwei et al. (2010) Wild-type and brachyolmia-causing mutant TRPV4 channels respond directly to stretch force. J Biol Chem 285:27176-81
Kung, Ching; Martinac, Boris; Sukharev, Sergei (2010) Mechanosensitive channels in microbes. Annu Rev Microbiol 64:313-29
Su, Zhenwei; Zhou, Xinliang; Loukin, Stephen H et al. (2009) Mechanical force and cytoplasmic Ca(2+) activate yeast TRPY1 in parallel. J Membr Biol 227:141-50
Loukin, Stephen H; Su, Zhenwei; Kung, Ching (2009) Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids. FEBS Lett 583:754-8
Martinac, Boris; Saimi, Yoshiro; Kung, Ching (2008) Ion channels in microbes. Physiol Rev 88:1449-90
Myers, Benjamin R; Saimi, Yoshiro; Julius, David et al. (2008) Multiple unbiased prospective screens identify TRP channels and their conserved gating elements. J Gen Physiol 132:481-6
Loukin, Stephen; Zhou, Xinliang; Kung, Ching et al. (2008) A genome-wide survey suggests an osmoprotective role for vacuolar Ca2+ release in cell wall-compromised yeast. FASEB J 22:2405-15
John Haynes, W; Zhou, Xin-Liang; Su, Zhen-Wei et al. (2008) Indole and other aromatic compounds activate the yeast TRPY1 channel. FEBS Lett 582:1514-8
Su, Zhenwei; Zhou, Xinliang; Haynes, W John et al. (2007) Yeast gain-of-function mutations reveal structure-function relationships conserved among different subfamilies of transient receptor potential channels. Proc Natl Acad Sci U S A 104:19607-12

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