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.

虽然视觉、嗅觉和味觉都是可以理解的，但在分子水平上， 不了解机械感觉，如触觉，听觉，血压，渗透压 平衡即使TRP超家族的离子通道被怀疑是 机械力转化为离子流，动物制剂的进展缓慢， 解剖学的复杂性、相关材料的缺乏和遗传学的繁琐。我们 将结合酵母分子遗传学的威力， 以及膜片钳技术在酵母TRP通道研究中的应用。 酵母TRP通道，Yvc1p，当细胞面对 高渗（=脱水），将Ca2+从液泡释放到细胞质中。下补片 钳夹时，Yvc1p通道被施加的膜拉伸力直接激活。 发现来自随机诱变后选择的yvcl突变体的通道保留了 它们的机械敏感性，但在其封闭或开放构象中不稳定。这些 突变原来涉及芳香残基，已知通常在 平行于脂双层的界面水平。界面位于带电的 脂质头部和脂质双层的疏水尾部，表面张力是 最高的似乎通道蛋白的芳香残基锚在细胞内， 蛋白质在这个水平上稳定其正常构象。我们假设， 拉伸力扰动双层内的力分布。该扰动 最终到达通道“门”（离子通路闭塞）通过芳香 主播与假设一致，外源添加的芳香族化合物 最近发现色氨酸、吲哚、对羟基苯甲酸酯等在体内激活Yvc1p 在膜片钳下。我们将继续生产氨基酸突变体 取代或缺失来定义力的机制和力所需的结构域 传输芳香族和两亲性化合物，其扰乱 双层将被系统地检查，以测试界面张力假说。

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