Preparation of artificial lipid bilayer membrane for molecular imaging of ion channels

Abstract

The cell membrane is a reaction field for material transport and signal transduction via membrane proteins. Among membrane proteins, ion channels play an important role in the regulation of ion concentrations and signal transduction in a living organism. Ion channels can maintain their correct conformation when encapsulated in lipid bilayers and most of them achieve their functions by assembling molecule in lipid bilayers. Therefore, experimental methods to evaluate their association states in membranes are useful for the development of expression methods of ion channels and elucidation of their drug effects. In this study, we aim to observe ion channel molecules by atomic force microscopy (AFM) using the supported lipid bilayer (SLB) system, which is an artificial cell membrane. Natural lipids extracts, such as asolectin, are used in cell-free membrane protein synthesis systems. However, they are a mixture of multi-component lipids, and thus it is difficult to fabricate uniform SLBs with good reproducibility. Because the outer membrane region of ion channels has a size of approximately 1 - 3 nm, it is necessary to fabricate a smooth SLB with a surface roughness of less than a nanometer order, to observe the molecular images of the ion channels. Therefore, we aimed to optimize the sample preparation and AFM observation suitable for the observation of the ion channels.

Vesicle suspensions were prepared by adding buffer (KCl 120 mM, HEPES 10 mM, pH 7.2/ KOH) to a vacuum-dry film of asolectin, which is a crude lipid from obtained from soybean, and a dye-labeled lipid (Rb-DPPE (Ex/Em: 560/583 nm)) at a ratio of 0.995:0.005 (w/w). SLBs were fabricated on mica substrates by the vesicle fusion method, and fluorescence microscopy, fluorescence recovery after photobleaching (FRAP) and AFM observations were performed in the buffer solution.

Fluorescence images showed that asolectin-SLB was formed, as uniform fluorescence intensity was observed on the mica substrate after the SLB formation operation. However, white bright spots (Fig. 1a, white arrows) and dark regions (Fig. 1a, black arrows) were also observed. These bright spots are unruptured adsorbed vesicles. Because the particle size of the vesicles is ~50 nm, the presence of adsorbed vesicles on the SLB may interfere with the observation of smaller size ion channels. The dark regions observed in the fluorescence images tended to appear more easily when the asolectin reagent was stored at -20 °C for more than 2 months, suggesting that they are intramembrane domains containing oxidized lipids. The presence of oxidized lipid domains is undesirable in ion channel observations because the composition and state of the lipid bilayer around membrane proteins can affect their activity. Reducing these adsorbed vesicles and domain regions is required for SLBs for molecular imaging by AFM. We investigated SLB preparation conditions by changing the temperature, time, and lipid concentration during the SLB formation process, as well as the storage method of asolectin reagent. As a result, SLBs with almost no adsorbed vesicles or dark domains were successfully prepared on a mica substrate (Fig. 1b). The roughness of the SLB surface observed by AFM was 0.69 nm (rms). This value is sufficiently smaller than the height of the outer membrane region of the ion channels (1 - 3 nm), and thus the asolectin SLB was suitable for their molecular imaging.