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

虽然视觉、嗅觉和味觉是可以理解的，但在分子水平上，我们 不了解机械感官，如触觉、听觉、血压、渗透压 平衡。即使色氨酸超家族的离子通道被怀疑能转导 机械力注入离子通量，动物制剂进展缓慢，因为 解剖结构的复杂性，相关材料的短缺，以及繁琐的遗传学。我们 将通过结合酵母分子遗传学的能力来补充动物工作 以及膜片钳技术在酵母色氨酸通道研究中的应用。 酵母Trp通道，Yvc1p，当细胞面对 高渗透压(=脱水)从液泡向细胞质释放钙离子。在补丁下 钳位，Yvc1p通道由施加的膜拉力直接激活。 随机诱变后选择的yvc1突变体的通道被发现保留了 它们的机械敏感性，但在闭合或开放构象中不稳定。这些 突变被证明涉及芳香族残基，已知的位置通常是 平行脂双分子层的界面水平。界面中心位于带电的 脂头和脂双层的疏水尾部，这是表面张力 最高的。似乎通道蛋白的芳香族残基锚定了 这一水平的蛋白质以稳定其正常构象。我们假设增加了 拉伸力扰乱了双层内的力分布。这种微扰 最终通过芳香族化合物到达通道“门”(离子通道闭塞) 锚。与假设一致，外源添加的芳香族化合物 (色氨酸、吲哚、对羟基苯甲酸酯等)最近被发现在体内激活Yvc1p 在膜片钳下。我们将继续产生含有氨基酸的突变体 替换或删除，以定义武力的机制和所需的领域 变速箱。芳香族和两亲性化合物扰乱了分子间的界面 为了验证界面张力假说，我们将对双分子层进行系统的研究。

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.