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

Abstract

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of welldefined 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. Video LinkThe video component of this article can be found at http://www.jove.com/video/50436/ IntroductionCells 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 the detrimental effects from the mutant proteins in human diseases.Lipids surround all membrane proteins integrated in bilayers 2,3 . In eukaryotic membranes, the various different types of lipids are known to be organized into microdomains 4, 5 . Many membrane proteins were shown to be distributed among these microdomains as well as the bulky fluid phase of membranes 3,6 . The mechanism underlying the organization of the microdomains and the delivery of membrane proteins into them and the physiological significance of such distributions are clearly important but remain poorly understood. One major technical difficulty in studying the lipid effects on membrane proteins is the reliable reconstitution of biochemically purified membrane proteins int...

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

“…Following expression and purification (Fig. S2A), the protein was reconstituted into liposomes containing a 3:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE):1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and applied to planar lipid bilayers of the same lipid composition for electrophysiological analysis following procedures established for other ion channels (21,22). Macroscopic currents recorded from bilayers containing BEST1 in the presence of ∼300 nM free Ca 2+ are shown in Fig.…”

“…Frozen liposomes were thawed and sonicated for approximately 10 s using an Ultrasonic Cleaner (Laboratory Supplies Company). All data are from recordings made using components analogous to the Warner planar lipid bilayer workstation (Warner Instruments) (21,22 ], ∼300 nM, as determined using Fura-2). Chlorided silver (Ag/AgCl) wires were used as electrodes, submerged in 3 M KCl, and connected to the bath solutions via agar-KCl salt bridges [2% (wt/vol) agar, 3 M KCl].…”

Novel organelle anion channels formed by chromogranin B drive normal granule maturation in endocrine cells

“…2C). We first tested if CHGB is a channel by bilayer recordings (Lee et al, 2013). The membrane-remodeling property made it difficult to record from many CHGB molecules.…”

bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

“…During membrane reconstitution, many factors, including temperature, rate of detergent removal, ionic strength, pH, etc., may affect bilayer formation (Lee et al, 2013). Our stepwise removal of detergents may control the fusion of small membrane patches around proteins, as well as, in the later phase, the fusion of small vesicles in the solution into the bilayers around the proteins.…”

“…Similarly, we experienced difficulty in introducing sphingolipids into solvent-based BLMs and found that 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in decane forms a sticky gel-like phase over time, probably caused by trace amounts of water. Additionally, the solvent-based BLMs have a significant amount of solvent (decane or squalene, for example), which may alter the physicochemical properties of the bilayers, especially phase separation, interactions between neutral lipids (such as cholesterol), and the islands of solvent molecules in membranes composed of composite lipids (Needham et al, 1988;Lee et al, 2013). To overcome many of these technical limitations, we developed a chemical procedure to generate a new bead-supported unilamellar membrane (bSUM) system.…”