Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan; Nanomaterials Research Division, Research Institute of Electronics, Shizuoka University, Shizuoka 422-8529, Japan; Department of Physics, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan.
Biochim Biophys Acta Biomembr. 2020 Oct 1;1862(10):183381. doi: 10.1016/j.bbamem.2020.183381. Epub 2020 Jun 3.
The effect of membrane potential on plasma membrane damage generated by antimicrobial peptides (AMPs) is an important, yet poorly characterized, process. Here, we studied the effect of membrane potential (φ) on pore formation by magainin 2 (Mag) in single giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. Various membrane potentials in GUVs containing gramicidin A were generated as a result of K concentration gradients. First, we examined Mag-generated membrane permeation of the water-soluble fluorescent probe calcein in single DOPG/DOPC-GUVs in the presence of membrane potential. The results indicate that the rate constant (k) of Mag-induced pore formation increased with increasing negative membrane potentials. Analysis of the rim intensity of single GUVs interacting with low concentrations of a fluorescent probe, carboxyfluorescein-labeled Mag (CF-Mag), using confocal laser scanning microscopy (CLSM) shows that the concentration of CF-Mag in the membrane greatly increased with negative membrane potentials. This indicates that the binding constant of CF-Mag to the membrane increased with more negative membrane potentials. To elucidate the location of Mag in a GUV with φ during Mag-induced pore formation, we examined the interaction of Mag and a low concentration of a CF-Mag mixture with single GUVs containing the water-soluble fluorescent probe AF647 using CLSM. The data indicate that CF-Mag locates in the external leaflet of single GUVs until just before pore formation. Based on these data, we conclude that the increase in the surface concentration of Mag is one of the primary causes of the increase in k with negative membrane potential.
膜电位对抗菌肽（AMPs）引起的质膜损伤的影响是一个重要但研究不足的过程。在这里，我们研究了膜电位（φ）对由两性霉素 2（Mag）在由二油酰基磷脂酰甘油（DOPG）/二油酰基磷脂酰胆碱（DOPC）组成的单个巨大单层囊泡（GUV）中形成孔的影响。含有短杆菌肽 A 的 GUV 中产生了各种膜电位，这是由于 K 浓度梯度的结果。首先，我们在存在膜电位的情况下，在含有膜电位的单个 DOPG/DOPC-GUV 中检查了 Mag 引起的水溶性荧光探针 calcein 的膜渗透。结果表明，Mag 诱导孔形成的速率常数（k）随负膜电位的增加而增加。使用共聚焦激光扫描显微镜（CLSM）分析与低浓度荧光探针羧基荧光素标记的 Mag（CF-Mag）相互作用的单个 GUV 的边缘强度表明，膜中 CF-Mag 的浓度随负膜电位的增加而大大增加。这表明 CF-Mag 与膜的结合常数随更负的膜电位而增加。为了阐明 Mag 在带 φ 的 GUV 中的位置在 Mag 诱导的孔形成过程中，我们使用 CLSM 检查了 Mag 和低浓度 CF-Mag 混合物与含有水溶性荧光探针 AF647 的单个 GUV 的相互作用。数据表明，CF-Mag 位于单个 GUV 的外叶层，直到孔形成之前。基于这些数据，我们得出结论，Mag 的表面浓度增加是 k 随负膜电位增加的主要原因之一。
