Format

Send to

Choose Destination
J Vis Exp. 2013 Jul 13;(77):e50436. doi: 10.3791/50436.

Reconstitution of a Kv channel into lipid membranes for structural and functional studies.

Author information

1
Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, USA.

Abstract

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

PMID:
23892292
PMCID:
PMC3735301
DOI:
10.3791/50436
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for MyJove Corporation Icon for PubMed Central
Loading ...
Support Center