Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri, USA.
Microcirculation. 2010 Nov;17(8):629-40. doi: 10.1111/j.1549-8719.2010.00059.x.
Changes in smooth muscle cell (SMC) membrane potential (Em) are critical to vasomotor responses. As a fluorescent indicator approach would lessen limitations of glass electrodes in contracting preparations, we aimed to develop a Forster (or fluorescence) resonance energy transfer (FRET)-based measurement for Em.
The FRET pair used in this study (donor CC2-DMPE [excitation 405 nm] and acceptor DisBAC(4) (3)) provide rapid measurements at a sensitivity not achievable with many ratiometric indicators. The method also combined measurement of changes in Ca(2+) (i) using fluo-4 and excitation at 490 nm.
After establishing loading conditions, a linear relationship was demonstrated between Em and fluorescence signal in FRET dye-loaded HEK cells held under voltage clamp. Over the voltage range from -70 to +30 mV, slope (of FRET signal vs. voltage, m) = 0.49 ± 0.07, r(2) = 0.96 ± 0.025. Similar data were obtained in cerebral artery SMCs, slope (m) = 0.30 ± 0.02, r(2) = 0.98 ± 0.02. Change in FRET emission ratio over the holding potential of -70 to +30 mV was 41.7 ± 4.9% for HEK cells and 30.0 ± 2.3% for arterial SMCs. The FRET signal was also shown to be modulated by KCl-induced depolarization in a concentration-dependent manner. Further, in isolated arterial SMCs, KCl-induced depolarization (60 mM) measurements occurred with increased fluo-4 fluorescence emission (62 ± 9%) and contraction (-27 ± 4.2%).
The data support the FRET-based approach for measuring changes in Em in arterial SMCs. Further, image-based measurements of Em can be combined with analysis of temporal changes in Ca(2+) (i) and contraction.
平滑肌细胞（SMC）膜电位（Em）的变化对血管舒缩反应至关重要。由于荧光指示剂方法可以减少玻璃电极在收缩制剂中的局限性，因此我们旨在开发一种基于Förster（或荧光）共振能量转移（FRET）的 Em 测量方法。
本研究中使用的 FRET 对（供体 CC2-DMPE[激发 405nm]和受体 DisBAC(4)(3)]）提供了快速测量，其灵敏度高于许多比率指示剂。该方法还结合了使用 fluo-4 测量 Ca(2+)（i）的变化和在 490nm 处激发。
在建立了加载条件后，在电压钳下保持的负载有 FRET 染料的 HEK 细胞中，证明了 Em 与荧光信号之间存在线性关系。在从-70 到+30mV 的电压范围内，斜率（FRET 信号与电压的关系，m）=0.49±0.07，r(2)=0.96±0.025。在大脑动脉 SMC 中也获得了类似的数据，斜率（m）=0.30±0.02，r(2)=0.98±0.02。在 HEK 细胞中，FRET 发射比率在-70 至+30mV 的保持电位下变化 41.7±4.9%，在动脉 SMC 中变化 30.0±2.3%。FRET 信号还表现出对 KCl 诱导去极化的浓度依赖性调制。此外，在分离的动脉 SMC 中，KCl 诱导的去极化（60mM）测量伴随着 fluo-4 荧光发射的增加（62±9%）和收缩（-27±4.2%）。
数据支持用于测量动脉 SMC 中 Em 变化的基于 FRET 的方法。此外，可以将基于图像的 Em 测量与 Ca(2+)（i）和收缩的时间变化分析结合起来。
