Improved Pressure-Clamp Technique for Studying Mechanosensitive Channels

9318255 McBride最近发展了一种结合膜片钳技术的压力钳技术(1)，用于研究单细胞和全细胞机械敏感(MS)通道电流的动力学特性。压力夹技术是基于平衡负压和正压，以在膜片上达到所需的压力。除了用于激活MS通道电流外，它还可以提供一种方便的方法来应用定义的和可重复的压力/吸气方案，以形成所有膜片钳实验所需的紧密密封。该项目的主要目标是改进现有的原型，使其成为研究MS通道的研究人员以及一般膜片钳人员都能接触到的方便单元。主要的改进集中在努力将夹具的响应时间从目前的10ms缩短到亚毫秒时间范围。另一项相关的改进涉及系统的小型化，这将使夹具更加紧凑和方便。该提案的另一个方面涉及在应用快速压力/吸力步骤期间确定膜运动的动态。例如，膜运动是否在MS通道激活之前，或者甚至是必要的？这将通过高分辨率(时间和空间)视频显微镜来解决。我们将同时监测压力/吸气步骤中的压力、电压、电流、膜电容和膜运动。伊，发展了一种与膜片钳技术相结合的压力钳技术(1)！！！F 3 Times新罗马符号&amp；Arial 1 Courier D“h E=抽象沙漠国王，BIR沙漠国王，BIR

9318255 McBride Recently, a pressure-clamp technique has been developed (1) which is used in conjunction with patch-clamp techniques to study the kinetic properties of single and whole-cell mechanosensitive (MS) channel currents. The pressure-clamp technique is based on the balancing of negative and positive pressures to achieve the desired pressure at the membrane patch. Apart from its use in the activation of MS channel currents it can also provide a convenient means of applying defined and reproducible pressure/suction protocols for tight seal formation necessary in all patch-clamp experiments. The main goals of this project are to improved the existing prototype and make it into a convenient unit accessible to researchers studying MS channels as well as to patch-clampers in general. The main improvements center around efforts to reduce the response time of the clamp from its present value of 10 ms to the sub-millisecond time range. Another related improvement involves miniaturization of the system which will make the clamp a more compact and convenient package. Another aspect of the proposal involves determining the dynamics of membrane movement during the application of rapid pressure/suction steps. For instance, does membrane movement precede or is it even necessary for MS channel activation? This will be addressed by high resolution (both temporal and spatial) video microscopy. We will simultaneously monitor the pressure, voltage, current, membrane capacitance and membrane movement during the pressure/suction step. y, a pressure-clamp technique has been developed (1) which is used in conjunction wit h patch-clamp tech ! ! ! F 3 Times New Roman Symbol & Arial 1 Courier D " h E E = abstract Deseree King, BIR Deseree King, BIR