25] Simplified fast pressure-clamp technique for studying mechanically gated channels

Abstract

A fundamental feature of the tight seal patch-clamp technique is the existence of a suction port in the patch pipette holder. This is critical for applying suction to the membrane patch to form a tight seal. This feature also enables, after seal formation, the application of pressure/ suction to stimulate the membrane patch mechanically. Initially, mouth-applied suction was used to obtain the seal and provide the stimulation. Although this is convenient, it lacks precision in terms of the magnitude and duration of the mechanical stimulus. This chapter describes a simpler but fast system known as “pressure clamps” and provides more details for its construction. The basic strategy of the pressure clamp is that the desired pressure applied to the patch pipette is achieved by a balancing of pressure and suction. Central to this balancing is the use of a proportional piezoelectric valve whose opening is proportional to the applied voltage. Through feedback control of this valve, the amount of pressurized N 2 allowed to enter a mixing chamber can be regulated to balance the constant outflow due a continual vacuum efflux and thus achieve the desired pressure.