Regulation of ion channel function by the host lipid bilayer examined by a stopped-flow spectrofluorometric assay.
Regulation of ion channel function by the host lipid bilayer examined by a stopped-flow spectrofluorometric assay.
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通过停流荧光分光光度法检测宿主脂质双层对离子通道功能的调节。
DOI：
10.1016/j.bpj.2014.01.027
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发表时间：
2014
影响因子：
3.4
通讯作者：
Andersen,OlafS
中科院分区：
文献类型：
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作者：
Rusinova,Radda;Kim,DorothyM;Nimigean,CrinaM;Andersen,OlafS
为了在特定的膜环境中检测配基门控离子通道的功能，我们开发了一种基于连续混合荧光的停流分析方法。使用通道渗透性猝灭剂(例如，铊，Tl+)来确定通道活性，该猝灭剂是包裹在大的单层小泡中的水溶性荧光团(8-氨基萘-1，3，6-三磺酸)，其中所述通道已被重建，这允许快速的溶液改变。为了验证该方法，我们探索了泡外质子(H+)对野生型KCSA通道及其非失活(E71A)KCSA突变体的激活作用。对于这两种类型的通道，重建产量的逐日变异性(从荧光猝灭的时间进程判断)为<；10%。E71AKCSA的激活曲线与单通道电生理的结果相似，野生型和E71AKCSA的激活曲线难以区分，表明通道的激活和失活是分开的过程。然后，我们研究了通过改变脂双层组成来调节KCSA的激活。增加酰基链长(从C18：1增加到C22：1)，但不增加形成双层磷脂混合物中POPG的摩尔分数(>；0.25)，改变KCSA H+门控。活化曲线的双层厚度依赖的位移暗示了表观H+亲和力和协作性的降低。对双层环境和时间分辨率的控制使该方法成为探索离子通道的配体激活和失活以及通道门控如何随着通道的脂双层环境或其他调节过程的变化而变化的有效方法。
To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl+) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H+). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C18:1to C22:1in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H+gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H+affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels' lipid bilayer environment or other regulatory processes.