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Abstract：
Subtle changes in the membrane potential of pulmonary arterial smooth muscle cells (PASMCs) are pivotal for controlling pulmonary vascular tone, e.g., for initiating Hypoxic Pulmonary Vasoconstriction, a vital mechanism of the pulmonary circulation. In our study, we evaluated the ability of the fluorescence resonance energy transfer (FRET)-based voltage-sensor Mermaid to detect such subtle changes in membrane potential. Mouse PASMCs were isolated and transduced with Mermaid-encoding lentiviral vectors before the acceptor/donor emission ratio was assessed via live cell FRET-imaging. Mermaid\'s sensitivity was tested by applying specific potassium chloride (KCl) concentrations. These KCl concentrations were previously validated by patch clamp recordings to induce depolarization with predefined amplitudes that physiologically occur in PASMCs. Mermaid\'s emission ratio dose-dependently increased upon depolarization with KCl. However, Mermaid formed unspecific intracellular aggregates, which limited the usefulness of this voltage sensor. When analyzing the membrane rim only to circumvent these unspecific signals, Mermaid was not suitable to resolve subtle changes in the membrane potential of ≤10 mV. In summary, we found Mermaid to be a suitable alternative for reliably detecting qualitative membrane voltage changes of more than 10 mV in primary mouse PASMCs. However, one should be aware of the limitations associated with this voltage sensor.
摘要：
肺动脉平滑肌细胞（PASMC）膜电位的细微变化对于控制肺血管张力至关重要，例如，用于启动低氧性肺血管收缩，肺循环的重要机制.在我们的研究中,我们评估了基于荧光共振能量转移（FRET）的电压传感器美人鱼检测膜电位这种细微变化的能力。分离小鼠PASMC并用编码美人鱼的慢病毒载体转导,然后通过活细胞FRET成像评估受体/供体发射比。通过应用特定的氯化钾(KCl)浓度来测试美人鱼的灵敏度。先前通过膜片钳记录验证了这些KCl浓度，以诱导具有生理上发生在PASMC中的预定义幅度的去极化。用KCl去极化时，美人鱼的发射比剂量依赖性增加。然而,美人鱼形成非特异性细胞内聚集体，这限制了这种电压传感器的实用性。当分析膜边缘只是为了规避这些非特定信号时，美人鱼不适用于解决≤10mV的膜电位的细微变化。总之,我们发现美人鱼是可靠检测原代小鼠PASMC中超过10mV的定性膜电压变化的合适替代方法。然而,人们应该意识到与这种电压传感器相关的限制。
