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

Abstract

The cellular resting membrane potential (Vm) 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 Vm, ideally over long periods of time. Here, we introduce two ratiometric genetically encoded Vm indicators, rArc and rASAP, and imaging and analysis procedures for measuring differences in average resting Vm 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 (KV10.1) syndrome on median resting Vm of HEK293T cells. Real-time long-term monitoring of Vm 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 Vm in health and disease.

An ultrasensitive genetically encoded voltage indicator uncovers the electrical activity of non-excitable cells

“…Fusion of mKate2 to a potentiometric GEVI is not only suited to determine the molecular brightness of a sensor molecule but also allows for absolute V m calibration, as we have shown previously (35). However, we observed that the fusion of mKate2 reduced the expression level of the sensor by a factor of 3.1 (Fig.…”

“…As a proof of concept, we recorded F 480 /F 400 from HEK293T cells stably expressing rEstus. We treated cells with 1 μM gramicidin, an ionophore that short-circuits the plasma membrane when integrated (35). The difference in F 480 /F 400 between control cells and those treated with gramicidin was readily apparent on a single-cell level (Fig.…”

“…While single-cell V m quantification was achieved with voltage-sensitive dyes using fluorescence-lifetime imaging (47), no GEVI-based approach offered sufficient resolution to quantify the V m of individual cells (35–38). We demonstrated that rEstus can be effectively calibrated using dual-excitation single-emission ratiometry.…”

“…Thus, the location of mid-potential (V half ) of the brightness–voltage relationship, which also defines the sensitivity optimum of the sensor, becomes highly relevant. In addition, measurements of slow changes in V m during, for example, the cell cycle or the quantitative comparison of V m in independent cell populations, require calibratable fluorescence signals that are independent of variations in the expression level (35,36). While several attempts by us and others were made to calibrate the signal of GEVIs using methods such as fluorescence lifetime imaging (37), pump-probe protocols (38), or dual-color ratiometry (35), none of these approaches achieved V m calibration at the single-cell level.…”

Harnessing the Membrane Potential to Combat Cancer Progression

“…Clearly, for such interventions to be effective at targeting the Vm, the wider context of tissue-, organ-wide and long-range endogenous electric fields needs to be considered 40 , as well as the possibility that cancer cells in certain tumor types may function as an electrical syncytium via gapjunctional communication 41 . A barrier to progression in this area remains the absence of reliable methods to accurately quantify small Vm changes in vitro and in vivo, although encouraging progress is now being made in this regard 42,43 . In addition, further work is required to address whether the Vm-dependent effects reported thus far are generalisable across different tumor types.…”