Imaging single cardiac ryanodine receptor Ca2+ fluxes in lipid bilayers.
Imaging single cardiac ryanodine receptor Ca2+ fluxes in lipid bilayers.
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DOI：
10.1016/s0006-3495(04)74091-6
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发表时间：
2004
影响因子：
3.4
通讯作者：
S. Peng;N. Publicover;G. Kargacin;D. Duan;J. Airey;J. Sutko
中科院分区：
文献类型：
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作者：
S. Peng;N. Publicover;G. Kargacin;D. Duan;J. Airey;J. Sutko
在此和随附的报告中，我们描述了两个步骤，单通道成像和通道固定化，必要的使用光学成像来分析兰尼碱受体（RyR）通道的功能重建在脂质双层。一个光学双层系统能够激光扫描共聚焦成像的fluo-3荧光由于Ca 2+通量通过单一RyR 2通道和单通道电流的同时记录。设计了一种电压命令协议，其中通过单个RyR 2通道的Ca 2+通量的幅度、时间过程、形状以及因此的量仅由跨双层施加的电压控制。使用该系统、电压命令方案和导致饱和RyR 2 Ca 2+电流的Ca 2+浓度（25- 50 mM），与具有0.25-14 pA幅度的Ca 2+电流同时记录成比例的fluo-3荧光。Ca 2+火花，类似于那些获得与传统的显微镜为基础的激光扫描共聚焦系统，在小鼠心室心肌细胞使用光学双层系统成像。光学双层膜的效用如何细胞外在的RyR 2通道，如钙缓冲和扩散，改变荧光-3荧光响应RyR 2钙电流，并解决其他当前的研究问题进行了讨论。
In this and an accompanying report we describe two steps, single-channel imaging and channel immobilization, necessary for using optical imaging to analyze the function of ryanodine receptor (RyR) channels reconstituted in lipid bilayers. An optical bilayer system capable of laser scanning confocal imaging of fluo-3 fluorescence due to Ca2+flux through single RyR2 channels and simultaneous recording of single channel currents was developed. A voltage command protocol was devised in which the amplitude, time course, shape, and hence the quantity of Ca2+flux through a single RyR2 channel is controlled solely by the voltage imposed across the bilayer. Using this system, the voltage command protocol, and concentrations of Ca2+(25–50mM) that result in saturating RyR2 Ca2+currents, proportional fluo-3 fluorescence was recorded simultaneously with Ca2+currents having amplitudes of 0.25–14 pA. Ca2+sparks, similar to those obtained with conventional microscope-based laser scanning confocal systems, were imaged in mouse ventricular cardiomyocytes using the optical bilayer system. The utility of the optical bilayer for systematic investigation of how cellular factors extrinsic to the RyR2 channel, such as Ca2+buffers and diffusion, alter fluo-3 fluorescent responses to RyR2 Ca2+currents, and for addressing other current research questions is discussed.