Characterization of Horizontal Lipid Bilayers as a Model System to Study Lipid Phase Separation

Abstract

Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range D(lat) = 10-25 mum(2)/s and rotational diffusion coefficients ranging from D(rot) = 2.8 - 1.4 x 10(7) s(-1) were measured in pure liquid-disordered (L(d)) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (L(o)) domains segregated from the L(d) phase at 23 degrees C. The lateral mobility of lipids in L(o) domains was around eightfold lower compared to those in the L(d) phase, whereas the rotational mobility decreased by a factor of 1.5. Burst-integrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the L(o) phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion-channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from L(o) domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the L(d) phase.

Lipid nanodomains change ion channel function

“…1b). A previous study of gramicidin inserted into planar bilayers of a DOPC/distearoylphosphatidylcholine/CHL lipid raft mixture 23 demonstrated only a single lifetime at temperatures below the miscibility transition, and simultaneous fluorescence microscopy in this study confirmed that the gramicidin is excluded from the DSPC-rich domains. However, when gramicidin is introduced into planar bilayers of a 1/1/1 mixture of DOPC/SPM/CHL, channels of relatively long duration (slow channels) are mixed with (fast) channels of much shorter duration (Fig.…”

Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS)

“…For Atto647N-DOPE, we measured [36,37], and that, vice-versa, the non-radiative decay rate k nr = k S1 -k fl is only sensitive to the environment, i.e. to the rate of collisions and the polarity of collision partners, we estimated , which correspond well to values measured in black lipid bilayers far away from the glass surface [38]. Figure 5 shows the calculated three-dimensional brightness profile when detecting the emission of Atto647 fluorophores dissolved in PBS buffer.…”

Heterogeneous rotational diffusion of a fluorescent probe in lipid monolayers

“…Heterogeneous dynamics has been previously reported for lipid diffusion in membrane systems 42–45 . The two experimental demonstrations include a measure of steady-state single molecule anisotropy distributions of fluorescent probes in the liquid-ordered state of ternary lipid mixtures 42 and polymer lateral diffusion in a supported lipid bilayer in the disordered phase 44 .…”