Abstract
Recent studies have demonstrated that annular lipids can directly change the voltage-gating of voltage-gated ion channels. We recognized that it was a challenge to insert membrane proteins into a bilayer that mimics the eukaryotic cell membranes in current model systems, especially the membranes that contain sphingomyelin and cholesterol. A new system is needed to overcome this technical barrier. In this study we developed a stable unilamellar vesicle system that is supported by unidirectionally inserted membrane proteins, and offers the capability of controlling lipid composition with relative ease. A voltage-gated potassium channel, KvAP, was used as a model system, and was selectively anchored onto the surface of micron-sized beads. Our data suggested that with a high surface density of channel molecules, it was feasible to introduce various lipids and form continuous membranes around the beads. The unilamellar nature of the bilayers was demonstrated by cryo-electron microscopic observations, and the functionality of the channels in the bead-buttressed unilamellar membrane (bBUM) system was examined by electrical recordings of voltage-gated activities. We anticipate that this novel membrane system will provide a new technique to study how lipids influence membrane proteins.
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