Single-Channel Recording of Ligand-Gated Ion Channels

Abstract

Single-channel recordings reveal the microscopic properties of individual ligand-gated ion channels. Such recordings contain much more information than measurements of ensemble behavior and can yield structural and functional information about the receptors that participate in fast synaptic transmission in the brain. With a little care, a standard patch-clamp electrophysiology setup can be adapted for single-channel recording in a matter of hours. Thenceforth, it is a realistic aim to record single-molecule activity with microsecond resolution from arbitrary cell types, including cell lines and neurons.

Figure 1

Recording configurations in mammalian cells. (A) Excising an inside-out patch from the cell-attached configuration often results in a bleb, from which no recording can be made. The outer surface can be removed by brief exposure of the patch to air. Outside-out patches form within the pipette from the resealing of the patch membrane after a smooth and assertive withdrawal of the patch pipette from the whole-cell configuration. For an organellar patch, the plasma membrane is sliced and the organelle of interest (preferably labeled; here shown as a black oval) is pushed out through the incision. Agonist molecules (black) are shown in the pipette or bath solution, as appropriate. The outer leaflet of the plasma membrane is shown in black to aid orientation. (B) A typical recording (30 sec) is shown for the rat glycine α1 receptor in cell-attached mode. Chloride-conducting openings are upward at negative membrane potential (pipette potential was approximately +70 mV in this recording). The bar under the trace indicates the magnified section (shown at bottom) illustrating a cluster with high open probability.

Figure 2

The single-channel recording landscape. The larger the conductance, or the slower the kinetics, the easier it is to obtain recordings, because the signal-to-noise ratio is larger at a given filter setting. The size of each circle corresponds to the approximate number of single-channel papers for the channel in question in the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) in 2014. The range is from ~5 (HCN) to ~600 (muscle nicotinic). Abbreviations: VGSC, voltage-gated sodium channel; VGPC, voltage-gated proton channel; VGCC, voltage-gated calcium channel; HCN, hyperpolarization-gated cation channel; CNG, cyclic nucleotide-gated channel.