Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

Abstract

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment. Electroformation is an efficient mean to produce giant liposomes >10 μm in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory. We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Regulation of TRPV1 Ion Channel by Phosphoinositide (4,5)-Bisphosphate

“…However, whether PI(4,5)P 2 activates or inhibits TRPV1 has been debated for over a decade and remains unresolved (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). As an activator, PI(4,5)P 2 has been proposed to associate with TRPV1 under basal conditions, acting as a cofactor for channel activation.…”

Characterizing the Structure and Interactions of Model Lipid Membranes Using Electrophysiology

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Enzymosomes: A Rising Effectual Tool for Targeted Drug Delivery System

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