Improved Pressure-Clamp Technique for Studying Mechanosensitive Channels

最近，一种压力钳技术被开发出来(1)，它与膜片钳技术一起用于研究单个和全细胞机械敏感（MS）通道电流的动力学特性。压力钳技术是基于平衡负压和正压来达到膜贴片所需的压力。除了用于激活MS通道电流外，它还可以为所有膜片钳实验中必要的紧密密封形成提供一种方便的方法，用于应用定义和可重复的压力/吸力协议。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的贴片钳的方便单元。主要的改进集中在努力将钳的响应时间从目前的10毫秒减少到亚毫秒的时间范围。另一个相关的改进涉及系统的小型化，这将使夹具更紧凑和方便的包装。该方案的另一个方面涉及在应用快速压力/吸入步骤期间确定膜运动的动力学。例如，膜运动是否先于质谱通道激活，或者是否必要？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、电压、电流、膜电容和膜在压力/吸入步骤中的运动。y,一种压力钳技术已经被开发出来(1)，它与膜片钳技术一起使用​!!!F 3 Times New Roman Symbol & Arial 1 Courier（信使）D " h E E = abstract Deseree King, BIR Deseree King， 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