Passive Translocation of Phospholipids in Asymmetric Model Membranes: Solid-State 1H NMR Characterization of Flip–Flop Kinetics Using Deuterated Sphingomyelin and Phosphatidylcholine,Langmuir

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Passive Translocation of Phospholipids in Asymmetric Model Membranes: Solid-State 1H NMR Characterization of Flip–Flop Kinetics Using Deuterated Sphingomyelin and Phosphatidylcholine
Langmuir ( IF 3.7 ) Pub Date : 2023-09-20 , DOI: 10.1021/acs.langmuir.3c01650
Hirofumi Watanabe ₁ , Shinya Hanashima _{1,

2} , Yo Yano ₁ , Tomokazu Yasuda _{1,

3} , Michio Murata _{1,

3}

Affiliation

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, (CH₃)₃N⁺, in the choline headgroup of N-palmitoyl-sphingomyelin (PSM) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) gave rise to a relatively sharp signal in magic-angle spinning solid-state ¹H NMR (MAS-ss-¹H 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 Pr³⁺ to the medium surrounding aLUVs selectively shifted the chemical shift of the (CH₃)₃N⁺ 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^–6 s^–1, 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.

中文翻译：

非对称模型膜中磷脂的被动易位：使用氘代鞘磷脂和磷脂酰胆碱对触发器动力学进行固态 1H NMR 表征

尽管细胞膜中的横向和小叶间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是未知的。在这里，我们建立了一种确定不对称大单层囊泡（aLUV）的外叶和内叶之间磷脂分配比的方法。N-棕榈酰-鞘磷脂 (PSM) 和 1,2-二油酰-sn-甘油-3-磷脂酰胆碱 (DOPC)的胆碱头基中的三甲基铵基团 (CH ₃ ) ₃N ⁺在魔角旋转固态¹ H NMR (MAS- ss - ¹ H NMR)。PSM 和 DOPC 具有相同的头基结构，但通过氘化另一种磷脂的三甲基铵基团选择性地观察到一种磷脂。^{将 Pr 3+}添加到 aLUV 周围的介质中，选择性地将外部小叶中的 (CH ₃ ) ₃N ⁺基团的化学位移与内部小叶中的化学位移相转移，这使得可以估计未标记的小叶间分布比aLUV 中的脂质。使用这种方法，我们评估了PSM和DOPC在含胆固醇aLUV的外叶和内叶之间的易位，在aLUV制备后，PSM和DOPC主要分别分布在外叶和内叶中；它们的翻转率和翻转率分别约为 2.7 和 6.4 × 10 ^–6 s ^–1。在 aLUV 的被动对称化过程中，由于膜顺序的变化，脂质易位率降低，可能是通过形成注册的液体有序域。与对称 LUV 的结果比较表明，脂质不对称可能不会显着影响脂质易位速率，而横向脂质-脂质相互作用可能是这些条件下脂质易位的主导因素。这些发现强调了在评估细胞膜中磷脂的不对称性时考虑同一小叶内横向脂质相互作用对脂质翻转率的影响的重要性。

更新日期：2023-09-20

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