Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Summary

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

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.

Introduction

Cells exchange materials and information with their environment through the functions of specific membrane proteins ¹. Membrane proteins in cell membranes function as pumps, channels, receptors, intramembrane enzymes, linkers and structural supporters across membranes. Mutations that affect the membrane proteins have been related to many human diseases. In fact, many membrane proteins have been the primary drug targets because they are important and easily accessible in cell membranes. It is therefore very important to understand the structure and function of various membrane proteins in membranes, and make it possible to devise novel methods to alleviate t....

Protocol

1. Expression and Purification of KvAP Channel (Figure 1)

1. Preparation Work - Day 0
   1. Rinse the glass flasks for the bacterial culture with deionized water (diH₂O) and MilliQ H₂O (MQH₂O) to remove trace of detergent from general dishwashing.
   2. Autoclave 1,000 ml LB medium in 2.8 L Erlenmeyer flasks (total two-liter culture as an example here). Low hardness of the water was found to be important for the successful culture of the transformed bacteria.
   3. Autoclave 100 ml LB medium in 500 ml flasks
   4. Transform 60 μl of XL1-Blue competent cells with 200 ng of the pQE60 plas....

Results

The general flow of the experiments for purifying the KvAP channel into biochemical homogeneity is described in Figure 1A. Typical samples during the expression and purification of the protein is showed in the SDS-PAGE gel in Figure 1B. The protein after the IMAC purification is relatively pure. The yield of the KvAP channel is about 1.0 mg/Liter culture.

Solubilization of lipid vesicles with detergents needs to be worked out for each pair of lipid vs. deterge.......

Discussion

The reconstitution of the KvAP channels into different membranes has been used in several studies ^8-10. Following the idea of ensuring the distribution of lipids between detergent/lipid mixed micelles and the protein/detergent/lipid mixed micelles, we are able to reach nearly complete reconstitution of the KvAP into membranes made of very different lipids. Each tetrameric KvAP channel needs ~100 lipid molecules to fully cover its transmembrane domain. The essential requirement is to allow enough lipid molecule.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptone	RPI Corp.	T60060
Yeast Extract	RPI Corp.	Y20020
NaCl	Fisher	S271-3
Tris Base	RPI Corp.	T60040
Potassium Chloride	Fisher	BP366-500
n-Dodecyl-β-D-Maltoside	Affymetrix	D322S	Sol-grade
n-Octyl-β-D-Glucoside	Affymetrix	O311	Ana-grade
Aprotinin	RPI Corp.	A20550-0.05
Leupeptin	RPI Corp.	L22035-0.025
Pepstatin A	RPI Corp.	P30100-0.025
PMSF	SIGMA	P7626
Dnase I	Roche	13407000
Bio-Bead SM-2	Bio-Rad	152-3920
HEPES	RPI Corp.	H75030
POPE	Avanti Polar Lipids	850757C
POPG	Avanti Polar Lipids	840457C
DOGS	Avanti Polar Lipids	870314C
DMPC	Avanti Polar Lipids	850345C
Biotin-DOPE	Avanti Polar Lipids	870282C
DOTAP	Avanti Polar Lipids	890890C
NeutrAvidin agarose beads	Piercenet	29200
Dialysis Tubing	Spectrum Laboratories, Inc	132-570
Pentane	Fisher	R399-1
Decane	TCI America	D0011
MTS-PEG5000	Toronto Research Cemicals	M266501