Unité Mixte de Recherche 168, Physico-Chimie Curie, Centre National de la Recherche Scientifique, Institut Curie, Centre de Recherche, Université Pierre et Marie Curie, Paris, France.
PLoS One. 2011;6(10):e25529. doi: 10.1371/journal.pone.0025529. Epub 2011 Oct 6.
Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid composition and physical state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimental settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane composition, tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositions and buffers containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs was characterized with quantitative confocal microscopy, and the protein density of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability.
电压门控离子通道是细胞兴奋性的关键因素。最近的研究表明，它们的行为可能强烈依赖于膜脂质组成和物理状态。由于膜性质在活细胞中被主动调节，并且在实验条件下难以控制，因此对膜/通道和通道/通道相互作用的体内研究具有挑战性。我们开发了一种将功能型电压门控离子通道重构到细胞大小的巨大单层囊泡（GUV）中的方法，其中可以控制膜组成、张力和几何形状。首先，纯化、荧光标记并重构电压门控钾通道 KvAP 到小的脂蛋白体中。然后通过电形成将小的脂蛋白体转化为 GUV。可以使用不同的脂质组成和含有低（5 mM）或接近生理（100 mM）盐浓度的缓冲液来形成 GUV。使用定量共焦显微镜对 GUV 中的蛋白掺入进行了表征，并且 GUV 的蛋白密度与它们形成的小脂蛋白体相当。此外，膜片钳测量证实了重构的通道保留了钾选择性和电压门控激活。含有功能型电压门控离子通道的 GUV 将允许在控制膜状态的同时研究通道活性、分布和扩散，并且应该证明是理解膜如何调节细胞兴奋性的有力工具。
