AFM Of Voltage And Mechanically Gated Channels

描述：这项工作的长期目标是了解细胞是如何 从机械和电气输入产生和传输信号。更多 具体地说，这项建议使用以下方法检查离子通道的属性 同步原子力显微镜(AFM)、膜片钳电生理学和 钙离子显像法。该提案还涉及上述方法的发展。 与在纳米制造领域扩大能力相结合 技术。电力和机械力的耦合将是 以细胞膜和特定离子通道为特征。 机械门控通道。机械敏感离子通道的激活是 由膜张力的变化所带来。在真核细胞中， 分布在细胞外基质、双层和 细胞骨架。这些系统表现出依赖于时间的松弛，这掩盖了 激活通道的力。原子力显微镜做得很好 机械刺激器，能够在已知距离上产生已知的力 微观维度。当原子力显微镜与膜片钳结合时，它是 有可能将电池的机械和电学特性联系起来。时间 依赖点扫描将表征应力松弛速率、力-体积 将使用源自合规性、机械性的对比度来创建图像 转导电流和局部钙离子的变化。定义物理属性的步骤 与传导相关，刺激应力、应变和速度是不同的。 为了确定特定的细胞外基质元素、悬臂的作用 使用特定的配基来拉动这些元素上的已知方向 用原子力显微镜。实验将测试局部变形是否会改变双层 或者它是否表现为一种全球流体。其他实验将测量 渗透膨胀的全球压力如何影响这些特性。克隆的 带有反应性基团的机械敏感通道将单独连接到 作为局部应变的函数测量悬臂梁和通道门控。 电压门控通道。对中四移动的估计完全在 原子力显微镜的分辨率。AFM悬臂将连接到克隆的半胱氨酸 带有带有马来酰亚胺或其他巯基的连接子的突变型K+通道 试剂。使用当前的门控协议，实验将测量距离 渠道中的不同站点可以移动。这些运动将是相关的 具有门控电流和沟道开口。两者之间的等价性 电动力和机械力将通过比较 移动的距离是电压和机械力的函数。当地人 S4周围的电场将由电力产生的力来测量 作为S4已知位置的价态的函数。

DESCRIPTION: The long-range goal of this work is to understand how cells generate and transmit signals from mechanical and electrical inputs. More specifically, this proposal examines the properties of ion channels using simultaneous atomic force microscopy (AFM), patch clamp electrophysiology and Ca2+ imaging. The proposal also deals with method developments in the above areas and in nanofabrication to expand the capabilities of the combined technique. The coupling of electrical and mechanical forces will be characterized in cell membranes and in specific ion channels. Mechanically gated channels. The activation of mechanosensitive ion channels is brought about by changes in membrane tension. In eukaryotic cells, the forces are distributed between the extracellular matrix, the bilayer, and the cytoskeleton. These systems display time-dependent relaxations that obscure the force that activates the channels. The atomic force microscope makes a fine mechanical stimulator capable of producing known forces over known distances at microscopic dimensions. When the AFM is combined with a patch-clamp, it is possible to correlate the cell's mechanical and electrical properties. Time dependent point scans will characterize stress relaxation rates, force-volume images will be created using contrast derived from compliance, mechanical transduction currents and changes in local Ca2+. To define physical properties relevant for transduction, stimulation stress, strain and velocity are varied. To establish the role of particular extracellular matrix elements, cantilevers with specific ligands are used to pull in known directions on these elements with the AFM. Experiments will test whether local deformation changes bilayer tension or whether it behaves as a global fluid. Other experiments will measure how global stress of osmotic swelling influences these properties. Cloned mechanosensitive channels with reactive groups will be individually linked to the cantilever and the channel gating measured as a function of local strain. Voltage gated channels. The estimates of S4 movement are well within the resolution of the AFM. The AFM cantilever will be connected to cloned, cysteine mutant, K+ channels that with linkers bearing maleimide or other sulfhydryl reagents. Using gating current protocols, experiments will measure the distance that different sites in the channel can move. The movement will be correlated with gating currents and channel opening. The equivalence between the electrical forces and the mechanical forces will be measured by comparing the distances moved as a function of voltage and mechanical force. The local electrical field around S4 will be measured by the electrically generated force as a function of the valence at known positions of S4.