Perforated Whole-Cell Patch-Clamp Recording

Abstract

Perforated whole-cell patch-clamp is a variant of the patch-clamp technique used to measure the sum activity of ion channels in the plasma membrane of a single cell. Its defining feature is that electrical access to the cell is obtained through inclusion of a pore-forming antibiotic in the patch pipette which perforates the sealed patch of membrane in contact with the patch pipette. The antibiotic pores allow equilibration of small monovalent ions between the patch pipette and the cytosol whilst maintaining endogenous levels of divalent ions such as Ca²⁺ and signalling molecules such as cAMP. Therefore, the perforated patch-clamp technique is ideal for studying ion channels whilst maintaining the integrity of second messenger signalling cascades. Other benefits of using perforated patch-clamp over conventional patch-clamp include reduced current rundown and stable whole-cell recording lasting >1 h. In this chapter, the application of the perforated patch-clamp technique for the study of heterologously expressed Kv7 potassium channels will be discussed in detail including benefits and limitations of the technique.