Patch Clamped Giant Unilamellar Vesicles Containing Reconstituted Ion Channels

Abstract

There is increasing interest in how the chemical and physical properties of lipid membranes affect the function of mammalian ion channels. As evidenced by recent work on KvAP and Kv1.2, as well as studies of the physical nature of patches themselves, these effects are both intriguing and difficult to study. For the TRP family of ion channels these questions are especially important as membrane lipids, such as phosphoinositide (4,5) bisphosphate (PIP2), play an active role in regulating their functional properties. Although much work has been done to address the mechanism by which lipids regulate TRP channels in intact cells and excised patches, the physical interactions that govern channel regulation are still unknown. We report here on our efforts to establish high resolution control over membrane chemical composition and physical properties. We find that giant unilammelar vesicles can be synthesized with desired lipid compositions and subsequently studied using patch-clamp techniques. Reconstitution of functional TRP channels into these synthetic lipid membranes would provide a well-controlled experimental paradigm for studying the function and mechanism of channel-lipid interactions.