AFM Of Voltage And Mechanically Gated Channels

电压和机械门控通道的 AFM

• 批准号: 6520163
• 负责人: FREDERICK SACHS
• 金额: $ 30.22万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520163
• 关键词: atomic force microscopy; biological signal transduction; biomechanics; biophysics; calcium channel; cell membrane; cysteine; electrophysiology; eukaryote; light microscopy; lipid bilayer membrane; membrane transport proteins; method development; nanotechnology; potassium channel; sensory mechanism; site directed mutagenesis; stimulus /response; tissue /cell culture; viscosity; voltage /patch clamp; voltage gated channel

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.

描述：这项工作的长期目标是了解细胞如何 从机械和电气输入生成并传输信号。更多的 具体来说，该提案使用以下方法检查离子通道的特性 同时原子力显微镜 (AFM)、膜片钳电生理学和 Ca2+ 成像。该提案还涉及上述方法的开发 领域和纳米制造，以扩大联合的能力 技术。电力和机械力的耦合将是 其特征在于细胞膜和特定离子通道。 机械门控通道。机械敏感离子通道的激活是 由膜张力的变化引起。在真核细胞中，力 分布在细胞外基质、双层和 细胞骨架。这些系统显示出与时间相关的松弛，从而掩盖了 激活通道的力量。原子力显微镜可以很好地观察 能够在已知距离上产生已知力的机械刺激器 微观尺寸。当 AFM 与膜片钳结合使用时， 可以将电池的机械和电气特性关联起来。时间 相关点扫描将表征应力松弛率、力体积 图像将使用源自顺应性、机械性的对比度来创建 传导电流和局部 Ca2+ 的变化。定义物理属性 与转导相关的刺激应力、应变和速度各不相同。 确定特定细胞外基质元素、悬臂梁的作用 具有特定配体的用于在这些元素上沿已知方向拉动 与原子力显微镜。实验将测试局部变形是否改变双层 张力或它是否表现为整体流体。其他实验将测量 渗透膨胀的全局压力如何影响这些特性。克隆 具有反应基团的机械敏感通道将单独连接到 悬臂梁和通道选通作为局部应变的函数进行测量。 电压门控通道。 S4运动的估计完全在 AFM 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接头的突变体 K+ 通道 试剂。使用门控电流协议，实验将测量距离 频道中的不同站点可以移动。运动将是相关的 具有门控电流和通道开放。之间的等价性 电力和机械力将通过比较来测量 作为电压和机械力的函数移动的距离。当地的 S4 周围的电场将通过电产生的力来测量 作为 S4 已知位置的化合价的函数。