Diffusion of single cardiac ryanodine receptors in lipid bilayers is decreased by annexin 12.
Diffusion of single cardiac ryanodine receptors in lipid bilayers is decreased by annexin 12.
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DOI：
10.1016/s0006-3495(04)74092-8
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发表时间：
2004
影响因子：
3.4
通讯作者：
S. Peng;N. Publicover;J. Airey;J. Hall;H. Haigler;D. Jiang;S. Chen;J. Sutko
中科院分区：
文献类型：
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作者：
S. Peng;N. Publicover;J. Airey;J. Hall;H. Haigler;D. Jiang;S. Chen;J. Sutko
表征了心脏兰尼碱受体 (RyR2) 在脂质双层中的扩散。 RyR2 位置通过成像 Fluo-3 荧光（由于 Ca2+ 流经 RyR2 通道）或来自与 Alexa 488 缀合或含有绿色荧光蛋白的 RyR2 的荧光来监测。记录单通道电流以确保研究功能通道。 RyR2 的表观扩散系数 (DRyR) 为 1.2×10−8cm2s−1，平均路径长度为 5.0μm。最佳使用光学方法分析 RyR2 通道功能需要限制 RyR2 扩散。因此，我们测试了膜联蛋白 12 的作用，它以 Ca2+ 依赖性方式与阴离子磷脂相互作用。将膜联蛋白 12 (0.25–4.0μM) 添加到含有 80:20 比例的磷脂酰乙醇胺/磷脂酰丝氨酸的双层的反面，以浓度依赖性方式减少 RyR2 扩散。膜联蛋白 12 (2μM) 使表观 DRyR683 倍从 1.2–10−8 减少到 1.8×10−11cm2s−1，平均路径长度减少 10 倍，从 5.0 减少到 0.5μm，而天然双层的电导或 Ca2+ 或苏拉明对 RyR2 通道的激活没有明显变化。因此，膜联蛋白 12 可能为优化脂质双层中 RyR2 通道的光学分析提供有用的工具。
Diffusion of cardiac ryanodine receptors (RyR2) in lipid bilayers was characterized. RyR2 location was monitored by imaging fluo-3 fluorescence due to Ca2+flux through RyR2 channels or fluorescence from RyR2 conjugated with Alexa 488 or containing green fluorescent protein. Single channel currents were recorded to ensure that functional channels were studied. RyR2 exhibited an apparent diffusion coefficient (DRyR) of 1.2×10−8cm2s−1and a mean path length of 5.0μm. Optimal use of optical methods for analysis of RyR2 channel function requires that RyR2 diffusion be limited. Therefore, we tested the effect of annexin 12, which interacts with anionic phospholipids in a Ca2+-dependent manner. Addition of annexin 12 (0.25–4.0μM) to thetransside of bilayers containing an 80:20 ratio of phosphatidylethanolamine/phosphatidylserine decreased RyR2 diffusion in a concentration-dependent manner. Annexin 12 (2μM) decreased the apparent DRyR683-fold from 1.2–10−8to 1.8×10−11cm2s−1and the mean path length 10-fold from 5.0 to 0.5μm without obvious changes in the conductance of the native bilayer or in activation of RyR2 channels by Ca2+or suramin. Thus, annexin 12 may provide a useful tool for optimizing optical analysis of RyR2 channels in lipid bilayers.