Optical determination of absolute membrane potential

Background

 

Membrane potential (V_mem) is an essential component of cellular physiology and is an essential signaling cue of cellular processes such as migration and cellular division. V_mem states in cell populations are largely uncharacterized due to the difficulty of patch clamp at specific stages but reports suggest that V_memplays a role in phases of the cell cycle^1,2. Although patch clamp electrophysiology is traditionally used for measuring V_mem,this method is highly invasive and time consuming. The possibility of modifying the signals under study with introduction of pipette solution into the cell and the low throughput of patch clamping created the need for alternative methods for determining absolute membrane potential^3,4.

 

Key findings

Lazzari-Deanet al have developed an interesting new method for optically quantifying absolute membrane potential in living cells using a fluorescent lifetime-based approach with single cell resolution. Lifetime-voltage relationships were established using VoltageFluor-fluorescent lifetime images (VF-FLIM) and simultaneous electrophysiology reporting a linear relationship for absolute V_memand a 20-fold improved accuracy over previous optical techniques. Single cell or cell populations can be measured, increasing spatial resolution 100-fold compared to patch clamping.

Evaluation of VF-FILM across cell lines

VF-FILM has high sensitivity (3.1 – 3.7ps/mV) and short average 0mV lifetime (1.78 – 1.87 ns). VF-FILM can be used across an array of cell types commonly used in electrophysiological studies (Fig 1) and can reliably be used in cell populations requiring only single-point calibration. All cells showed linear relationship between VF fluorescent lifetime and V_mem with a voltage resolution of 5mV or better. Not only does this mean a higher throughput, but also an increased spatial resolution compared to patch clamp.

 

Figure 1. VF-FLIM is a general and portable method for optically determining membrane potential. VF2.1.Cl lifetime-voltage relationships were determined with whole cell voltage clamp electrophysiology in five cell lines. (A) Slope and (B) 0 mV reference point of linear fits for the lifetime-voltage relationship, shown as mean ± S.E.M. Gray dots are single cells. (C) Representative lifetime-intensity overlay images for each cell line with the indicated cells (white arrow) held at -80 mV (top) or +80 mV (bottom). Lifetime scales are in ns. Scale bar is 20 μm. Reproduced from Figure 2. of the preprint

 

Membrane potential dynamics in epidermal growth factor signaling

The authors next used VF-FILM to elucidate the role of V_memduring EGF/EGF receptor (EGFR)-mediated signaling in A431 cells. EGF treatment results in a 15mV hyperpolarization within 60-90 seconds in approximately 80% of cells followed by a return to baseline in 15 minutes (Fig 2). Inhibition of EGFR and ErbB2 tyrosine kinase activity with the covalent inhibitor canertinib abolishes hyperpolarization. Additionally, blockade of the EGFR kinase domain with gefitnib also diminishes hyperpolarization indicating that A431 cells exhibit an EGF-induced hyperpolarization which is dependent on the kinase activity of EGFR.

 

Figure. 2 EGFR-mediated receptor tyrosine kinase activity produces a transient hyperpolarization in A431 cells. (A) Quantification of images. Vehicle (Veh.)/EGF added at black arrow. (B) Aggregated responses for various trials of cells treated with vehicle or EGF. (C) Quantification of images with Veh/EGF. (D) Average response of cells. (E) Lifetime images of A431 cells before and after EGF addition, with 500 nM canertinib (top) or 10 μM gefitinib (bottom). (F) Voltage changes 2.5 minutes after EGF addition in cells treated with DMSO (vehicle control) or an EGFR inhibitor. Scale bar is 20 μm. (C,F,H): Asterisks indicate significant differences between vehicle and EGF at that time point. (F): Asterisks reflect significant differences between EGF-induced voltage responses with DMSO vehicle or an EGFR inhibitor (n.s. p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, two-tailed, unpaired, unequal variances t-test). Reproduced from Figure 4. of the preprint

 

The authors identified the outward potassium channels as the mediating current of EGF-induced hyperpolarization. Block of K⁺ driving force with high extracellular [K⁺] completely abolished the EGF-induced hyperpolarization response whereas blockage of voltage-gated K⁺channels with 4-aminopyridine (4-AP) enhances the hyperpolarization. Specific inhibition of intermediate-conductance Ca²⁺-activated potassium channel K_Ca3.1 using TRAM-43 also abolished EGF-induced hyperpolarization. The hyperpolarizing current was found to be carried by K⁺ions passed through Ca²⁺-activated K⁺channel K_Ca3.1 and mediated by intracellular Ca²⁺stores. In the context of receptor tyrosine kinase signalling, V_mem may modulate the driving force for external Ca²⁺entry and act as a regulator for Ca²⁺signalling. Therefore, VF-FLIM could be a useful tool in assessing effects of small V_memchanges on signaling pathways in non-excitable cells.

 

Figure 3.EGF-induced hyperpolarization is mediated by a Ca activated K channel. (A) Comparison of the Vmem change 2.5 minutes after EGF addition in cells incubated in unmodified imaging buffer (HBSS) or in modified solutions. (B) Lifetime images of A431 cells treated with 4-AP or CTX. (C) Model for membrane hyperpolarization following EGFR activation. Scale bar is 20 μm. Bars are mean ± SEM. Asterisks reflect significant differences in EGF- stimulated Vmem change between the unmodified control (HBSS or DMSO) and modified solutions (n.s. p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, two-tailed, unpaired, unequal variances t-test). Reproduced from Figure 5. of the preprint

 

Why I liked this preprint

This is a nice article that shows in a simple way that membrane potential can be determined using VF-FLIM but that it can also be used to probe membrane potential in cellular states that are not accessible or challenging with standard electrophysiological techniques. The authors make a real effort to validate their method through several experiments and provide plenty of figures in the supplementary data to get a real understanding of the probe and its uses.

 

Questions to authors 

1) Cells in culture or in primary tissue are electrically connected via gap junctions, however VF-FLIM was found to be robust in small groups of cells. How many cells can be used in a population type recording and what are the spatial limitations of the technique?

2) In a long-term experiment how do you control for probe stability over time and exclude ion channel run down?

3) Is there a temperature dependency of the fluorescence of VF-FLIM?

 

References

1. Ouadid-ahidouch, H., Bourhis, X. Le & Roudbaraki, M. Changes in the K + current-density of MCF-7 cells during progression through the cell cycle : Possible Involvement of a h-ether . a-gogo K + channel. (2001).
2. Byrd, R. C. & Sciences, H. Changes in Membrane Potential During the Progression of MCF-7 Human Mammary Tumor Cells Through the Cell Cycle. 185,177–185 (1995).
3. Flag, T., Oh, L.-, Gst, T. & Web, W. Letters To Nature. Nature408,381–386 (2000).
4. Horn, R. & Korn, S. J. Prevention of rundown in electrophysiological recording. Methods Enzymol.207,149–155 (1992).

 

Tags: cell contraction, cell cycle, membrane potential

doi: https://doi.org/10.1242/prelights.9406

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