Membrane Mechanics, Osmotic Stress, and the Yeast Stretch-Activated TRP

膜力学、渗透压和酵母拉伸激活的 TRP

基本信息

批准号：
7730165
负责人：
YOSHIRO SAIMI
金额：
$ 31.19万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-05-01 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7730165
关键词：
American Amino Acid Substitution Amino Acids Animals Aromatic Amino Acids Aromatic Compounds Attention Automobile Driving Bacteria Bacteriology Biophysics Blood Pressure Cell membrane Cells Charge Chemicals Complement Complement component C1s Consultations Consumer Advocates in Research and Related Activities Cytoplasm Dehydration Electron Spin Resonance Spectroscopy Emergency Situation Engineering Equilibrium Escherichia coli Face Family Figs - dietary Funding Gated Ion Channel Genes Genetic Grant Harvest Head Health Hearing Heating Homologous Gene Human Indoles Invaded Ion Channel Ion Channel Gating Ions Laboratories Lipid Bilayers Lipids Location Mechanical Stimulation Mechanics Membrane Methods Modeling Molecular Molecular Conformation Molecular Genetics Mutagenesis Mutation Neck Parabens Pathway interactions Phenotype Play Preparation Process Proteins Recording of previous events Research Research Design Resolution Rest Role Sensory Site-Directed Mutagenesis Smell Perception Stress Stretching Structural Models Structure Surface Tension System TRP channel Tail Taste Perception Testing Time Touch sensation Tryptophan United States National Institutes of Health Vacuole Vision Work Yeasts antimicrobial attenuation base carbonyl group gain of function hydroxybenzoate improved in vivo interest interfacial member mutant patch clamp positional cloning public health relevance reconstitution research study simulation solute transmission process vibration yeast genetics

项目摘要

Although vision, olfaction and taste are understood, at the level of molecules, we do not understand mechanical senses such as touch, hearing, blood pressure, osmotic balance. Even when ion channels of the TRP superfamily are suspected to transduce mechanical force into ion flux, progress has been slow with animal preparations because of anatomical complexity, shortage of relevant materials, and cumbersome genetics. We will complement the animal work by combining the prowess of yeast molecular genetics and the resolution of patch clamp on the study of a TRP channel in yeast. The yeast TRP channel, Yvc1p, instantly opens when cells are confronted with hyperosmolarity (= dehydration) to release Ca2+ from vacuole to cytoplasm. Under patch clamp, Yvc1p channels are directly activated by applied membrane stretch force. Channels from yvcl mutants selected after a random mutagenesis are found to retain their mechanosensitivity but are unstable in their closed Or open conformations. These mutations turn out to involve aromatic residues, known to often in locations that parallel the lipid bilayer's interfacial level. The interface centers between the charged lipid head and the hydrophobic tail of the lipid bilayer, a point where surface tension is the highest. It appears that the aromatic residues of the channel protein anchor the protein at this level to stabilize its normal conformations. We hypothesize that added stretch force perturbs the force distribution within the bilayer. This perturbation eventually reaches the channel "gate" (the ion pathway occlusion) through the aromatic anchors. Consistent with the hypothesis, exogenously added aromatic compounds (tryptophan, indole, parabens, etc) have recently been found to activate Yvc1p in vivo and under patch clamp. We will continue to generate mutants with amino-acid substitutions or deletions to define the mechanism of and the domains required for force transmission. Aromatic and amphipathic compounds that perturb the interfaces of the bilayer will be systematically examined to test the interface-tension hypothesis.

尽管视觉，嗅觉和味道是在分子水平上被理解的，但我们不了解触摸，听力，血压，渗透等机械感官平衡。即使怀疑TRP超家族的离子通道也会传播机械力进入离子通量，动物制剂的进展缓慢，因为解剖学复杂性，相关材料的短缺和繁琐的遗传学。我们将通过结合酵母分子遗传学的能力来补充动物的工作以及在研究酵母中TRP通道的研究中的斑块夹的分辨率。酵母TRP通道YVC1P，当细胞面对面时立即打开高渗透度（=脱水）从液泡到细胞质释放Ca2+。在补丁下夹具，YVC1P通道通过施加的膜弹性直接激活。发现从随机诱变后选择的YVCL突变体的通道可保留它们的机械敏感性，但在其封闭或开放构象中不稳定。这些事实证明突变涉及芳香残留物，通常在地点已知平行脂质双层的界面水平。接口中心是在充电之间脂质头和脂质双层的疏水尾巴，表面张力为最高。看来通道蛋白的芳族残基锚定该水平的蛋白质可以稳定其正常构象。我们假设这添加了伸展力使双层中的力分布散布。这种扰动最终通过芳香族到达通道“门”（离子途径遮挡）锚。与假设一致，外源添加的芳香化合物（色氨酸，吲哚，对羟基苯甲酸酯等）最近被发现激活YVC1P体内并在斑块夹下。我们将继续产生具有氨基酸的突变体替换或删除以定义力的机理和域所需的域传播。芳香和两亲性化合物，扰动界面的界面将对双层进行系统检查以检验界面张力假设。