Characterization of Horizontal Lipid Bilayers as a Model System to Study Lipid Phase Separation
Characterization of Horizontal Lipid Bilayers as a Model System to Study Lipid Phase Separation
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DOI：
10.1016/j.bpj.2010.03.033
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发表时间：
2010-06-16
影响因子：
3.4
通讯作者：
Wagner, Richard
中科院分区：
文献类型：
--
作者：
Honigmann, Alf;Walter, Claudius;Wagner, Richard
人工类脂膜是一种利用电生理技术研究单个离子通道活性的模型系统。在这项研究中，我们利用单分子荧光涨落和电生理技术表征了人工双层体系的性质，并对其脂相分离动力学进行了研究。我们用共聚焦时间分辨各向异性来探测膜的微观粘度，测量了荧光标记的脂类在纳秒时间尺度上的旋转运动。同时，利用荧光相关光谱研究了脂类的横向扩散对其长程迁移率的影响。根据所用溶剂的不同，在纯液相无序(L-d)膜中测得的横向扩散系数范围为D-lat=10-25µm(2)/S，旋转扩散系数范围为D-RoT=2.8-1.4×10(7)S(-1)。在含有饱和和不饱和磷脂和胆固醇的三元混合物中，在23℃下，液体有序(L-o)结构域从L-d相分离。与L-d相相比，L-o结构域中脂质的横向迁移率降低了约8倍，而旋转迁移率降低了1.5倍。突发积分稳态各向异性直方图以及各向异性成像被用来可视化相分离双层中脂质探针的旋转迁移率。这些实验和在不同焦距下的荧光相关光谱测量表明，L-o相是一个不均匀的微环境。最后，我们展示了光电装置的潜力，以研究脂结构域对离子通道电生理性质的影响。我们发现甘草素A(Ga)的电生理活性与脂结构域的分配有关。在液-液相分离过程中，Ga在很大程度上被排除在L-O域之外。同时，由于L-d相中Ga的表面密度增加，电活性Ga晚餐的数量增加。
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 mu m(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 dinners increased due to the increased surface density of gA in the L-d phase.