Reconstitution of Voltage-Activated Potassium Channel into Phospholipid Bilayer

Abstract

Voltage-activated ion channels are expressed in the membranes of excitable cells, and open and close in response to changes in membrane voltage. Physiologically, they play diverse roles, from the generation of action potentials to triggering the release of neurotransmitters at synapses. To study the biochemical properties, these ion channels are usually purified and studied under detergent-solubilized condition, but many ion channels behave differently in detergent when compared to their native membrane environment. One recently introduced technique to overcome this issue is using lipid bilayer nanodiscs. Nanodiscs are a few nanometer-sized hockey-puck-shaped lipid bilayers solubilized by engineered apolipoprotein, membrane scaffold protein (MSP). MSP is comprised of short repeats of amphipathic alpha helies that surround the hydrophobic acyl chains of phospholipids. To investigate the structure and biochemical properties of voltage-activated potassium (Kv) channels in a membrane environment, we reconstituted a Kv channel into phospholipid bilayer nanodiscs using phospholipid and MSP. We first attempted to assemble empty nandiscs (nanodisc without Kv) to determine the optimal ratio between lipid and MSP that provides the best yields. Using these conditions, we reconstituted Kv channels into nanodiscs, and show that they elute as a single peak in gel-filtration chromatography. SDS-PAGE result indicates that the preparation contains each subunit corresponding to the Kv and MSP, confirming successful reconstitution of the Kv into nanodiscs. We also show that a voltage-sensor toxin can bind to the Kv channel in nanodiscs, confirming that the architecture of the Kv channel is well maintained in nanodiscs.