Digitonin does not flip across cholesterol-poor membranes
Digitonin does not flip across cholesterol-poor membranes
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DOI：
10.1016/j.jcis.2017.05.034
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发表时间：
2017-10-15
影响因子：
9.9
通讯作者：
Heerklotz，Heiko
中科院分区：
文献类型：
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作者：
Fan，Helen Y.;Heerklotz，Heiko
洋地黄苷常用于渗透细胞膜和溶解膜组分。它与膜上的胆固醇特异性地相互作用，从而导致气孔的形成。到目前为止，洋地黄苷与膜相互作用的机制仅定性描述。我们结合等温滴定量热法、动态光散射和zeta电位测量，在含有少量或不含胆固醇的模型膜中研究了这种相互作用。洋地黄苷在20°C时完全不对称地分裂成大的单层磷脂（PC）脂质囊泡（仅保留在外层小叶中），其分配系数为0.22±0.04 mM−1，Δ分配H为23.3±1.6 kJ mol−1。在外小叶中，洋地黄皂苷/脂质比超过0.1时，洋地黄皂苷胶束与囊泡共存而不溶解它们-即使在高洋地黄皂苷浓度下。在磷酸丝氨酸（PS）、PC/PS和PC/PS/胆固醇囊泡中也观察到洋地黄苷的这种“滞留”。当膜含有少量或不含胆固醇与20-30 mol%胆固醇时，洋地黄苷干扰和溶解膜的机制是非常不同的。因此，在细胞过程和膜蛋白研究的制备方案和实验设计中，应仔细考虑洋地黄苷的作用。
Digitonin is commonly used to permeabilize cell membranes and solubilize membrane components. It interacts specifically with cholesterol in the membrane which leads to the formation of pores. Thus far, the mechanism by which digitonin interacts with the membrane has only been described qualitatively. We investigated this interaction in model membranes that contain little or no cholesterol with a combination of isothermal titration calorimetry, dynamic light scattering, and zeta potential measurements. Digitonin partitions fully asymmetrically into large unilamellar vesicles of phosphocholine (PC) lipid at 20° C (remaining in the outer leaflet only), with a partition coefficient of 0.22±0.04 mM− 1 and Δ H of partitioning of 23.3±1.6 kJ mol− 1. Beyond a digitonin/lipid ratio of∼ 0.1 in the outer leaflet, digitonin micelles coexist with vesicles without solubilizing them—even at high digitonin concentrations. This “staying out” of digitonin was also observed with phosphoserine (PS), PC/PS, and PC/PS/cholesterol vesicles. The mechanism by which digitonin perturbs and solubilizes the membrane is very different when the membrane contains little or no cholesterol as opposed to 20–30 mol% cholesterol. The role of digitonin should thus be carefully considered in the design of preparative protocols and experiments in studies of cellular processes and membrane proteins.