Abstract
In vitro reconstitution of a membrane protein is the ultimate tool for understanding the molecular details of functional properties inherent to the isolated protein. In addition, reconstitution into pure synthetic lipid bilayers of defined composition is prerequisite to study of how membrane protein function may depend on local or bulk lipid properties. Unfortunately, in vitro reconstitution of large mammalian membrane proteins remains difficult to master. Using the TRPV1 ion channel, we have carried out an exhaustive optimization of the many steps involved in reconstitution into pure lipid vesicles. Optimization required techniques and analysis of data from a wide variety of disciplines: membrane protein purification and analysis; physical chemistry of lipids and detergent mixtures; lipid vesicle characterization using light scattering, sedimentation, and gel filtration chromatography; and, in our case, electrophysiology. We have integrated our results with those of others to develop a systematic strategy for reconstituting functional membrane proteins. Using patch-clamp analysis of TRPV1 in giant unilamellar vesicles, we will show how our strategy leads to consistent, reproducible recordings of channel currents and provides improved understanding of the effects of PIP2 on TRPV1 function.
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