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The cellular resting membrane potential ( V  m ) 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  m , ideally over long periods of time. Here, we introduce two ratiometric genetically encoded  V   m   indicators, rArc and rASAP, and imaging and analysis procedures for measuring differences in average resting  V  m  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 (K V 10.1) syndrome on median resting  V  m  of HEK293T cells. Real-time long-term monitoring of  V  m  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   m   in health and disease.

Monitoring of compound resting membrane potentials of cell cultures with ratiometric genetically encoded voltage indicators