Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, D-07745, Jena, Germany.
Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA.
Commun Biol. 2021 Oct 7;4(1):1164. doi: 10.1038/s42003-021-02675-0.
The cellular resting membrane potential (V) not only determines electrical responsiveness of excitable cells but also plays pivotal roles in non-excitable cells, mediating membrane transport, cell-cycle progression, and tumorigenesis. Studying these processes requires estimation of V, ideally over long periods of time. Here, we introduce two ratiometric genetically encoded V indicators, rArc and rASAP, and imaging and analysis procedures for measuring differences in average resting V between cell groups. We investigated the influence of ectopic expression of K channels and their disease-causing mutations involved in Andersen-Tawil (Kir2.1) and Temple-Baraitser (K10.1) syndrome on median resting V of HEK293T cells. Real-time long-term monitoring of V changes allowed to estimate a 40-50 min latency from induction of transcription to functional Kir2.1 channels in HEK293T cells. The presented methodology is readily implemented with standard fluorescence microscopes and offers deeper insights into the role of the resting V in health and disease.
细胞静息膜电位（V）不仅决定了可兴奋细胞的电响应性，而且在非兴奋细胞中也起着关键作用，介导膜转运、细胞周期进程和肿瘤发生。研究这些过程需要估计 V，理想情况下是在很长一段时间内。在这里，我们介绍了两种比率基因编码的 V 指示剂 rArc 和 rASAP，以及用于测量细胞群体之间平均静息 V 差异的成像和分析程序。我们研究了在外源表达 K 通道及其与 Andersen-Tawil（Kir2.1）和 Temple-Baraitser（K10.1）综合征相关的致病突变对 HEK293T 细胞中静息 V 中位数的影响。实时长期监测 V 的变化，使我们能够估计在 HEK293T 细胞中转录诱导到功能性 Kir2.1 通道的潜伏期为 40-50 分钟。所提出的方法很容易在标准荧光显微镜上实现，并为静息 V 在健康和疾病中的作用提供了更深入的了解。
