Recording of Ion Channel Activity in Planar Lipid Bilayer Experiments
Planar lipid bilayer is an electrophysiological technique that enables study of functional activities of ion channels, porins, and other pore-forming molecular complexes. The main purpose of this method is to monitor ion channels’ behavior at the single m
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Introduction Electrophysiological approaches are designed to evaluate physical properties and characteristics of ion channels. Planar lipid bilayer is one of the unique electrophysiological techniques that is intended to study specific channel properties of the purified complexes in a well-controlled artificial environment (1). The effectiveness of this method reveals the possibility to study and characterize ion channel behavior at the single molecule level. Furthermore, this technique is very useful for investigation of the direct effects of chemicals and enzymes on the channel while excluding possible indirect effects, which might be caused by the presence of regulatory proteins of the native membranes. Planar lipid bilayer experiments enable monitoring the transport rates of ions across membranes through incorporated ion channels. The experiments are performed in the chamber with cis- and transcompartments that are connected through a small aperture with a diameter ranging from 50 to 250 mm. A lipid solution is applied to the aperture with subsequent formation of a planar bilayer membrane on the hole. This is followed by reconstitution of ion channel proteins. Ion channels can be inserted into bilayer lipid
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_8, © Springer Science+Business Media, LLC 2013
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Eleonora Zakharian
membrane (BLM) directly from a micellar solution or fused with liposomes. After the channel is incorporated into the bilayer, the ionic current can be induced by applying a driving force. This force represents an electrochemical potential that has two components— electrical, which is called membrane potential (Vm or Dy) derived from charges or voltages applied across the membrane, and chemical component that derives from a chemical gradient with asymmetric ionic solutions. The ionic current through typical ion channel is measured in pico-amperes (pA), and potential or voltages are measured in millivolts (mV). Conductance of ions at a single-channel level is usually measured in a range of 10–1,000 pico-siemens (pS). Some ion channels such as ligand-gated channels would also require the presence of specific molecules-activators (ligands) in order to induce channel openings. In case of many mammalian channels this regulation can be quite complex and would require simultaneous presence of a number of molecular components and/or various physical factors (temperature, pressure) in order to stimulate channel openings. The advantage of planar lipid bilayer is that it increases likelihood to precisely identify all the chemical and physical factors that are required for, or supporting, the channel activity. The other benefits of this method include a possibility to alternate lipid composition or chemical compounds regulating channel activity that can be applied to either side of the membrane. Like any technique, planar lipid bilayer also has its disadvantages. Among the disadvantages of lipid bilayers are large c
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