Single channel recording is an extension of the patch-clamp technique described in the section Patch-clamp recording. Smaller area of membrane patch together with high resistance of gigaseal makes it possible to record individual openings of ionic channels present in the patch. The outside-out configuration of the patch-clamp technique is especially suitable for the study of chemically activated channels as the extracellular part of ion channels is exposed to the external solution and can be stimulated and modulated by drug application.

Principle:

The outside-out patch is made by first forming a high resistance cell-attached patch and then rupturing the membrane within the lumen of the pipette (see section Patch-clamp recording for details). When the whole-cell configuration is successfully formed, the pipette is then slowly pulled away from the cell. The part of membrane is also pulled away from the cell without losing high-resistance gigaseal connection between the glass and the membrane. Connection with the cell body narrows down until it spontaneously pinches off and membrane seals over the tip of the pipette. In this configuration the outside surface of the cell membrane is facing the bath and the cytoplasmic surface of the membrane is in contact with the solution inside the pipette. The patch potential is accurately controlled and single channel currents can be recorded from channels present in the membrane patch.

It is necessary to keep electrical noise of the whole setup as low as possible. Several measures to reduce noise usually include:

• proper electric grounding of the setup
• using Faraday cage and shielding to reduce pick-up of external electromagnetic signals
• using thick wall glass pipettes for making electrodes
• using Sylgard resin to improve noise parameters of microelectrodes
• keeping the pipette holder clean and dry
• high resistance gigaseal
• minimize resistance of solution/silver wire connection

Experiment schedule:

Pulling of microelectrodes

Covering of microelectrode by Sylgard and curing of resin by heat.

The whole cell configuration establishment - according to Chapter 5.

Pulling away of the microelectrode by the micromanipulator and forming outside out configuration.

Application of agonists and recording of current