Improved Pressure-Clamp Technique for Studying Mechanosensitive Channels

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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