Sofia University "St. Kliment Ohridski", Faculty of Biology, 8 Dragan Tzankov str., 1164 Sofia, Bulgaria; Medical University-Sofia, Faculty of Medicine, 1 Sv. Georgi Sofiiski Str., 1431 Sofia, Bulgaria.
Sofia University "St. Kliment Ohridski", Faculty of Biology, 8 Dragan Tzankov str., 1164 Sofia, Bulgaria.
Colloids Surf B Biointerfaces. 2020 May;189:110893. doi: 10.1016/j.colsurfb.2020.110893. Epub 2020 Feb 21.
Human bestrophin-1 (hBest1) is a transmembrane Ca- dependent anion channel, associated with the transport of Cl, HCO ions, γ-aminobutiric acid (GABA), glutamate (Glu), and regulation of retinal homeostasis. Its mutant forms cause retinal degenerative diseases, defined as Bestrophinopathies. Using both physicochemical - surface pressure/mean molecular area (π/A) isotherms, hysteresis, compressibility moduli of hBest1/sphingomyelin (SM) monolayers, Brewster angle microscopy (BAM) studies, and biological approaches - detergent membrane fractionation, Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine) and immunofluorescence staining of stably transfected MDCK-hBest1 and MDCK II cells, we report: 1) Ca, Glu and GABA interact with binary hBest1/SM monolayers at 35 °C, resulting in changes in hBest1 surface conformation, structure, self-organization and surface dynamics. The process of mixing in hBest1/SM monolayers is spontaneous and the effect of protein on binary films was defined as "fluidizing", hindering the phase-transition of monolayer from liquid-expanded to intermediate (LE-M) state; 2) in stably transfected MDCK-hBest1 cells, bestrophin-1 was distributed between detergent resistant (DRM) and detergent-soluble membranes (DSM) - up to 30 % and 70 %, respectively; in alive cells, hBest1 was visualized in both liquid-ordered (L) and liquid-disordered (L) fractions, quantifying protein association up to 35 % and 65 % with L and L. Our results indicate that the spontaneous miscibility of hBest1 and SM is a prerequisite to diverse protein interactions with membrane domains, different structural conformations and biological functions.
人源 bestrophin-1（hBest1）是一种跨膜 Ca2+ 依赖性阴离子通道，与 Cl-、HCO3- 离子、γ-氨基丁酸（GABA）、谷氨酸（Glu）的转运以及视网膜内环境稳态的调节有关。其突变形式可导致视网膜退行性疾病，被定义为 Bestrophinopathies。本研究采用物理化学 - 表面压力/平均分子面积（π/A）等温线、滞后、hBest1/神经鞘磷脂（SM）单层的可压缩性模量、偏光显微镜（BAM）研究以及生物学方法 - 去污剂膜分级、Laurdan（6-十二烷酰基-N，N-二甲基-2-萘胺）和稳定转染的 MDCK-hBest1 和 MDCK II 细胞的免疫荧光染色，报告：1）Ca2+、Glu 和 GABA 在 35°C 下与二元 hBest1/SM 单层相互作用，导致 hBest1 表面构象、结构、自组织和表面动力学发生变化。hBest1/SM 单层中混合的过程是自发的，蛋白质对双层膜的影响被定义为“增溶”，阻碍了单层从液体膨胀到中间（LE-M）状态的相变；2）在稳定转染的 MDCK-hBest1 细胞中，Bestrophin-1 分布在去污剂抗性（DRM）和去污剂可溶性膜（DSM）之间 - 分别为 30%和 70%；在活细胞中，hBest1 被可视化在液体有序（L）和液体无序（L）部分，定量蛋白质与 L 和 L 的关联高达 35%和 65%。我们的结果表明，hBest1 和 SM 的自发混合是蛋白质与膜域、不同结构构象和生物功能进行多种相互作用的前提。
