Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka 560-0043, Osaka, Japan.
Graduate School of Engineering, Tottori University, 4-101 Koyama-cho Minami, Tottori 680-8550, Japan.
Langmuir. 2023 Oct 31;39(43):15189-15199. doi: 10.1021/acs.langmuir.3c01650. Epub 2023 Sep 20.
Although lateral and inter-leaflet lipid-lipid interactions in cell membranes play roles in maintaining asymmetric lipid bilayers, the molecular basis of these interactions is largely unknown. Here, we established a method to determine the distribution ratio of phospholipids between the outer and inner leaflets of asymmetric large unilamellar vesicles (aLUVs). The trimethylammonium group, (CH), in the choline headgroup of -palmitoyl-sphingomyelin (PSM) and 1,2-dioleoyl--glycero-3-phosphatidylcholine (DOPC) gave rise to a relatively sharp signal in magic-angle spinning solid-state H NMR (MAS--H NMR). PSM and DOPC have the same headgroup structure, but one phospholipid was selectively observed by deuterating the trimethylammonium group of the other phospholipid. The addition of Pr to the medium surrounding aLUVs selectively shifted the chemical shift of the (CH) group in the outer leaflet from that in the inner leaflet, which allowed estimation of the inter-leaflet distribution ratio of the unlabeled lipid in aLUVs. Using this method, we evaluated the translocation of PSM and DOPC between the outer and inner leaflets of the cholesterol-containing aLUVs, with PSM and DOPC mostly distributed in the outer and inner leaflets, respectively, immediately after aLUV preparation; their flip and flop rates were approximately 2.7 and 6.4 × 10 s, respectively. During the passive symmetrization of aLUVs, the lipid translocation rate was decreased due to changes in the membrane order, probably through the formation of the registered liquid-ordered domains. Comparison of the result with that of symmetric LUVs revealed that lipid asymmetry may not significantly affect the lipid translocation rates, while the lateral lipid-lipid interaction may be a dominant factor in lipid translocation under these conditions. These findings highlight the importance of considering the effects of lateral lipid interactions within the same leaflet on lipid flip-flop rates when evaluating the asymmetry of phospholipids in the cell membrane.
虽然细胞膜中侧向和层间脂质-脂质相互作用在维持不对称脂质双层中发挥作用，但这些相互作用的分子基础在很大程度上尚不清楚。在这里，我们建立了一种确定不对称大单室脂质体（aLUV）中外层和内层之间磷脂分布比的方法。-棕榈酰鞘氨醇（PSM）和 1,2-二油酰基--甘油-3-磷酸胆碱（DOPC）的胆碱头部的三甲铵基团（CH）在魔角旋转固态 H NMR（MAS-H NMR）中产生相对尖锐的信号。PSM 和 DOPC 具有相同的头部结构，但通过氘化另一种磷脂的三甲铵基团可以选择性地观察到一种磷脂。向 aLUV 周围的介质中添加 Pr 会选择性地将外层（CH）基团的化学位移从内层的化学位移移动，这允许估计 aLUV 中未标记脂质的层间分布比。使用该方法，我们评估了 PSM 和 DOPC 在含有胆固醇的 aLUV 中外层和内层之间的易位，PSM 和 DOPC 主要分别分布在外层和内层，立即在 aLUV 制备后；它们的翻转和翻转速率分别约为 2.7 和 6.4×10 s。在 aLUV 的被动对称化过程中，由于膜有序性的变化，脂质易位速率降低，可能是通过形成注册的液体有序域。将结果与对称 LUV 的结果进行比较表明，脂质不对称性可能不会显著影响脂质易位速率，而侧向脂质-脂质相互作用可能是这些条件下脂质易位的主要因素。这些发现强调了在评估细胞膜中磷脂不对称性时，考虑同一层内侧向脂质相互作用对脂质翻转速率的影响的重要性。
