Howard Hughes Medical Institute and the Department of Neuroscience, University of Wisconsin, Madison, Wisconsin.
Howard Hughes Medical Institute and the Department of Neuroscience, University of Wisconsin, Madison, Wisconsin.
Biophys J. 2021 Apr 20;120(8):1367-1377. doi: 10.1016/j.bpj.2021.02.005. Epub 2021 Feb 12.
SNARE-mediated membrane fusion proceeds via the formation of a fusion pore. This intermediate structure is highly dynamic and can flicker between open and closed states. In cells, cholesterol has been reported to affect SNARE-mediated exocytosis and fusion pore dynamics. Here, we address the question of whether cholesterol directly affects the flickering rate of reconstituted fusion pores in vitro. These experiments were enabled by the recent development of a nanodisc⋅black lipid membrane recording system that monitors dynamic transitions between the open and closed states of nascent recombinant pores with submillisecond time resolution. The fusion pores formed between nanodiscs that bore the vesicular SNARE synaptobrevin 2 and black lipid membranes that harbored the target membrane SNAREs syntaxin 1A and SNAP-25B were markedly affected by cholesterol. These effects include strong reductions in flickering out of the open state, resulting in a significant increase in the open dwell-time. We attributed these effects to the known role of cholesterol in altering the elastic properties of lipid bilayers because manipulation of phospholipids to increase membrane stiffness mirrored the effects of cholesterol. In contrast to the observed effects on pore kinetics, cholesterol had no effect on the current that passed through individual pores and, hence, did not affect pore size. In conclusion, our results show that cholesterol dramatically stabilizes fusion pores in the open state by increasing membrane bending rigidity.
SNARE 介导的膜融合通过融合孔的形成进行。这种中间结构具有高度的动态性，可以在开放和关闭状态之间闪烁。在细胞中，胆固醇已被报道影响 SNARE 介导的胞吐作用和融合孔动力学。在这里，我们研究了胆固醇是否直接影响体外重建融合孔的闪烁率。这些实验得益于最近开发的纳米盘⋅黑脂膜记录系统，该系统能够以亚毫秒的时间分辨率监测新生重组孔在开放和关闭状态之间的动态转变。在纳米盘中形成的融合孔，这些纳米盘承载着囊泡 SNARE 突触融合蛋白 2，而黑脂膜则承载着靶膜 SNARE 突触融合蛋白 1A 和 SNAP-25B，明显受到胆固醇的影响。这些影响包括强烈减少从开放状态的闪烁，导致开放停留时间显著增加。我们将这些影响归因于胆固醇改变脂质双层弹性特性的已知作用，因为操纵磷脂以增加膜刚度反映了胆固醇的作用。与观察到的对孔动力学的影响相反，胆固醇对单个孔通过的电流没有影响，因此不会影响孔径。总之，我们的结果表明，胆固醇通过增加膜弯曲刚性，显著稳定开放状态下的融合孔。
