Zakany Florina, Szabo Mate, Batta Gyula, Kárpáti Levente, Mándity István M, Fülöp Péter, Varga Zoltan, Panyi Gyorgy, Nagy Peter, Kovacs Tamas
Division of Biophysics, Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Department of Genetics and Applied Microbiology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary.
Front Cell Dev Biol. 2021 Apr 12;9:647300. doi: 10.3389/fcell.2021.647300. eCollection 2021.
Although the largely positive intramembrane dipole potential (DP) may substantially influence the function of transmembrane proteins, its investigation is deeply hampered by the lack of measurement techniques suitable for high-throughput examination of living cells. Here, we describe a novel emission ratiometric flow cytometry method based on F66, a 3-hydroxiflavon derivative, and demonstrate that 6-ketocholestanol, cholesterol and 7-dehydrocholesterol, saturated stearic acid (SA) and ω-6 γ-linolenic acid (GLA) increase, while ω-3 α-linolenic acid (ALA) decreases the DP. These changes do not correlate with alterations in cell viability or membrane fluidity. Pretreatment with ALA counteracts, while SA or GLA enhances cholesterol-induced DP elevations. Furthermore, ALA (but not SA or GLA) increases endo-lysosomal escape of penetratin, a cell-penetrating peptide. In summary, we have developed a novel method to measure DP in large quantities of individual living cells and propose ALA as a physiological DP lowering agent facilitating cytoplasmic entry of penetratin.
尽管膜内偶极子电位（DP）总体呈阳性，可能会对跨膜蛋白的功能产生重大影响，但由于缺乏适用于活细胞高通量检测的测量技术，对其研究受到了严重阻碍。在此，我们描述了一种基于3-羟基黄酮衍生物F66的新型发射比率流式细胞术方法，并证明6-酮胆甾烷醇、胆固醇和7-脱氢胆固醇、饱和硬脂酸（SA）和ω-6γ-亚麻酸（GLA）会使DP升高，而ω-3α-亚麻酸（ALA）会使DP降低。这些变化与细胞活力或膜流动性的改变无关。用ALA预处理可抵消胆固醇诱导的DP升高，而SA或GLA则会增强这种升高。此外，ALA（而非SA或GLA）会增加穿膜肽（一种细胞穿透肽）的内吞溶酶体逃逸。总之，我们开发了一种在大量单个活细胞中测量DP的新方法，并提出ALA作为一种生理性DP降低剂，可促进穿膜肽进入细胞质。
