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

• 批准号: 7936078
• 负责人: CHING KUNG
• 金额: $ 32.32万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2011-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7936078
• 关键词: Amino Acid Substitution; Amino Acids; Animals; Aromatic Amino Acids; Aromatic Compounds; Attention; Automobile Driving; Bacteria; Bacteriology; Binding; Binding Sites; Biophysics; Blood Pressure; Cations; Cell membrane; Cells; Charge; Chemicals; Complement; Consultations; Cytoplasm; Dehydration; Electron Spin Resonance Spectroscopy; Elements; Emergency Situation; Engineering; Equilibrium; Escherichia coli; Face; Family; Figs - dietary; Future; Gated Ion Channel; Genetic; Hand; Harvest; Head; Health; Hearing; Heating; Homologous Gene; Housing; Human; In Vitro; Indoles; Invaded; Investigation; Ion Channel; Ion Channel Gating; Ion Channel Protein; Ions; Laboratories; Lead; Left; Lipid Bilayers; Lipids; Location; Mechanical Stimulation; Mechanics; Medical; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Molecular Genetics; Mutagenesis; Mutate; Mutation; Neck; Parabens; Pathway interactions; Phenotype; Play; Preparation; Process; Proteins; Recording of previous events; Research; Research Design; Resolution; Rest; Role; Sensory; Signal Transduction; Site; Site-Directed Mutagenesis; Smell Perception; Stimulus; Stress; Stretching; Structural Models; Structure; Surface Tension; System; TRP channel; Tail; Taste Perception; Testing; Touch sensation; Tryptophan; Vacuole; Vision; Voltage-Gated Potassium Channel; Work; Yeasts; antimicrobial; base; carbonyl group; fungus; hydroxybenzoate; improved; in vivo; interest; interfacial; member; mutant; patch clamp; positional cloning; public health relevance; reconstitution; research study; restoration; sensor; simulation; solute; stereochemistry; tool; transmission process; vibration; voltage; yeast genetics

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。

项目成果

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K+ channel.

羧基尾部形成一个离散的功能域，可阻止酵母 K 通道的关闭。

• DOI: 10.1073/pnas.042538599
• 发表时间: 2002
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Loukin,StephenH;Lin,Junyu;Athar,Umair;Palmer,Christopher;Saimi,Yoshiro
• 通讯作者: Saimi,Yoshiro