Voltage-gated channel proteins are activated by changes in membrane potential. A membrane potential can be simulated using spherical liposomes, by adjusting the buffer so that the internal and external ion concentrations are different. Voltage-gated channel proteins embedded in the liposome bi-layer function.
The aim of this project is to prepare liposomes that have a pH gradient across their lipid membrane, i.e., a different buffer and pH inside and outside. This will allow the membrane proteins embedded in the bilayer to be ‘captured in action’ in a close-to-native functional form by cryo-EM.
Cell membranes are comprised of a lipid bi-layer and proteins. They form a barrier between the cell interior and the surroundings. The different ion concentrations on either side of the membrane cause a potential difference across it known as the membrane potential (typically -40 to -80 mV).
The proteins embedded in cell membranes have various functions. Channel proteins span the whole membrane and regulate the transport of ions from the exterior to the interior of the cell and vice versa. Some do this passively by simply opening or shutting the channel, others actively promote the transport. Voltage-gated channel proteins are activated by a change in membrane potential, which causes them to switch from the closed to the open conformation or vice versa.
Voltage gated potassium channels are transmembrane protein complexes that form a pore specifically allowing the passage of potassium ions. One method to determine the structure of these and other membrane proteins is electron crystallography. For this, purified membrane proteins are mixed with lipids and induced to form two-dimensional crystals. These flat crystal sheets are then imaged by cryo-EM and analysed. There is no potential gradient across them as the protein is surrounded by the same buffer. The gradient required for voltage gated channel proteins to function can be created if they are embedded in a spherical lipid bilayer that encloses liquid, i.e., if they are embedded in the membrane of a liposome. The buffer conditions inside and outside the liposomes dictate whether they are in an open or a closed conformation.