This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.
Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.
Fluorescence-based, high-throughput activity assays of recombinant channel proteins have been highly effective in identifying small-molecule modulators that have the potential to become future therapies1,2,3,4. One common method of screening small molecule libraries for ion channel modulators is through measuring changes in membrane potential. Typically, these screens are conducted using recombinant channels expressed heterologously in cell lines such as HEK-293 cells5,6,7.
However, there is a need for validation of "hits" in relevant cell types. We propose that a secondary screen in relevant cell lines that endogenously express the channels of interest is desirable. Such a secondary screen will identify those modulators most likely to be effective in final validation assays that rely on less accessible primary human tissues.
There is also the need for assay systems that have the flexibility to report the activity of multiple ion channel targets in the same tissue type. For example, there is substantive published literature that supports the concept that CFTR and ENaC functionally interact8,9,10. Although several well-studied epithelial cell lines are known to express both CFTR and ENaC, to date, the assay conditions for studying both in a high-throughput manner have not been described.
This fluorescence-based membrane potential assay is versatile, as it employs an exogenous chemical probe to measure the function of CFTR and ENaC, bypassing the need for genetically encoded probes11. As proof of principle, two commonly used epithelial cell lines are studied as examples to highlight the critical steps necessary for measuring channel activity of both CFTR and ENaC.
1. Cell plating and differentiation
2. CFTR functional assay buffer solution preparation
3. CFTR functional assay
4. ENaC functional assay buffer solution preparation
5. ENaC functional assay
To measure CFTR channel activity, well-differentiated, adhered cells are incubated in a zero-chloride extracellular solution containing the membrane potential-sensitive dye. CFTR function is consistently detected as membrane depolarization after forskolin stimulation. Chloride efflux is detected as a sharp increase in fluorescence compared to the vehicle control. The fluorescence signal is sustained until the addition of CFTR inhibitor (CFTRInh172), leading to a rapid decline in the fluorescence intensity, as seen in Caco-2 cells (Figure 1A), and is reproducible in both Caco-2 and Calu-3 cells (Figure 1B). The assessment of CFTR activity as the difference in the maximal change in fluorescence after forskolin or the DMSO (vehicle). Individual points ranged between ± 3 standard deviations from the mean forskolin stimulation (black points) or DMSO control (grey points), which corresponded to a Z' factor of 0.586. This excellent score indicated the reproducibility of this assay (Figure 1C). In case there is a minor drift in the baseline, the impact of the drift can be minimized by extrapolating the baseline throughout the trace after activation up to the point of inhibitor addition. The response can be quantified as the area under the curve, with the lower limit defined by the extrapolated line.
ENaC activity can also be measured in confluent airway and colonic epithelial cells grown in 96-well plates. In contrast to the assay for CFTR channel activity, these cells are submerged in a high-sodium-containing solution, enabling sodium absorption through ENaC. With the acute addition of amiloride, sodium uptake is blocked, and cells experience relative membrane hyperpolarization. This is shown in Caco-2 cells at both low (10 µM) (Figure 2A) and high concentrations (50 µM) of amiloride (Figure 2A,B) and in Calu-3 cells with a high concentration of amiloride (50 µM) (Figure 2B). The ENaC assay was expanded to a 384-well plate format and demonstrated great reproducibility in ENaC inhibition. Similarly, the individual points ranged between ± 3 standard deviations from the mean amiloride inhibition (black points) or DMSO control (grey points). The ENaC response in Calu-3 cells reported a Z' factor of 0.629 with acute treatment using amiloride (50 µM) (Figure 2C), which indicates that these parameters support high-throughput screening.
Figure 1: CFTR channel measurement as changes in membrane depolarization in Caco-2 and Calu-3 cells. (A) Representative trace of CFTR stimulation with forskolin (1 µM)and DMSO control in Caco-2 cells. (B) Box and whisker plot of forskolin stimulation in Caco-2 and Calu-3 cells (**** P < 0.0001, n > 3 biological replicates, n > 3 technical replicates). Biological replicates are independent cell line passages; technical replicates refer to independent wells of the multiwell plates. Box plot depicts the median value; bounds depict IQR, with the whiskers defining the minima and maxima values. (C) Bland-Altman plot of reproducible CFTR response with forskolin stimulation compared to DMSO control in Caco-2 cells. Black points represent maximal absolute changes in fluorescence in response to forskolin stimulation. Grey points represent changes in fluorescence in response to DMSO control. Abbreviations: CFTR = Cystic Fibrosis Transmembrane Conductance Regulator; DMSO = dimethyl sulfoxide; IQR = interquartile range; Fsk = forskolin; ΔF/F0 = change in fluorescence. Please click here to view a larger version of this figure.
Figure 2: ENaC inhibition measurements through membrane hyperpolarization in Caco-2 and Calu-3 cells. (A) Representative trace of ENaC inhibition with amiloride (10 µM)relative to DMSO control in Caco-2 cells. (B) Box and whisker plot of amiloride inhibition in Caco-2 and Calu-3 cells (**** P < 0.0001, n > 3 biological replicates, n > 3 technical replicates). (C) Bland-Altman plot of reproducible ENaC response with amiloride inhibition compared to DMSO control in Calu-3 cells. Black points represent maximum absolute changes in fluorescence in response to amiloride inhibition. Grey points represent changes in fluorescence in response to DMSO control. Abbreviations: ENaC = epithelial sodium channel; DMSO = dimethyl sulfoxide; Amil. = amiloride; ΔF/F0 = change in fluorescence. Please click here to view a larger version of this figure.
Name | Concentration |
NMDG (N-Methyl-D-glucamine) | 150 mM |
Gluconic Acid Lactone | 150 mM |
Potassium Gluconate | 3 mM |
HEPES | 10 mM |
Table 1: Reagents and their corresponding concentrations for CFTR functional assay with zero sodium and chloride. Abbreviation: CFTR = Cystic Fibrosis Transmembrane Conductance Regulator.
Name | Concentration |
Sodium Gluconate | 150 mM |
Potassium Gluconate | 3 mM |
HEPES | 10 mM |
Table 2: Reagents and their corresponding concentrations for ENaC functional assay buffer with high sodium, zero chloride. Abbreviation: ENaC = Epithelial sodium (Na) Channel.
Here, we have described fluorescence-based methods for measuring CFTR and ENaC activity in the epithelial colorectal cancer cell line, Caco-2, and the human epithelial lung cancer cell line, Calu-3. These membrane potential assays in epithelial cell lines can be used to confirm the efficacy of small molecule modulator compounds, previously identified in heterologous expression systems, prior to final in vitro validation in primary patient-derived epithelial cultures.
It is imperative to confirm that the above measurements are appropriately attributed to the channel of interest. For example, the measurement ascribed to CFTR should show dependence on CFTR protein expression and the electrochemical driving force of chloride, which is modulated by forskolin and the inhibitor CFTRInh-172. Similarly, the membrane potential changes caused by 10 µM amiloride can be attributed to ENaC, if they are reliant on ENaC protein expression and an inward sodium driving force. Although we have confirmed these properties of ENaC in MDCK cells following transfection with all three subunits of ENaC2, we have not yet formally confirmed that the amiloride response measured in Caco-2 and Calu-3 cells is conferred by ENaC. This is particularly important, as amiloride can modulate the function of the sodium-proton exchanger NHE3 in addition to ENaC13. Conceivably, the amiloride-induced hyperpolarization observed in this assay could partially reflect the indirect effects of intracellular acidification on other channels. To confirm that the amiloride-induced hyperpolarization measured using this membrane potential-sensitive dye is conferred by ENaC in the above cell lines and more complex cell systems, such as iPSC-derived tissues, we are confirming that this activity is lost by knocking out an obligate ENaC subunit (beta) in these lines.
The success of these assays requires attention to several factors. First, the epithelial cultures must be confluent and well differentiated. For the measurement of CFTR and ENaC function, approximately 145,000 cells should be plated per well in 96-well plates or 45,000 cells per well in 384-well plates. Once the epithelial cells reach confluency (within 3-4 days from plating), allow 2-5 days of differentiation. This timing was determined previously based on maximal CFTR protein expression by immunoblotting14. Similarly, optimal timing was determined for functional ENaC expression in both cell lines, Calu-3 and Caco-2.
Nonconfluent monolayers or cell overgrowth leads to low reproducibility across technical replicates. In cases where cells in individual wells do not reach confluency at the same time, these plates should be discarded. Additionally, unstable baseline readings or lack of stimulation responses may be an indication of poor cell quality, which must be excluded from analysis. Reduced responses to the CFTR activator, forskolin, or the ENaC inhibitor, amiloride, could potentially reflect the use of excessively passaged cells15. Although not an issue for Calu-3 and Caco-2 cells, other cells or tissues may not adhere well to the plates and may require precoating of the wells. For example, in previous studies, 2D cholangiocyte cultures were attached to the plates using collagen coating16. Alternatively, the adherence of 2D intestinal tissues was increased using Poly-L-Lysine2.
Further, in testing the modulation of ion channels by small molecules using a fluorescence-based assay, it is critical that potential artifacts conferred by fluorescent properties of the small molecules be addressed. To confirm the specificity of functional responses, membrane potential assays can be further validated using conventional electrophysiological methods, such as Ussing studies17.
After confirming that the response detected by membrane potential-sensitive dyes is specifically conferred by the channel of interest, these assays have tremendous potential to validate promising modulators of epithelial ion channels in their relevant cellular context. This platform can bridge the gap between high-throughput modulator screens in heterologous expression systems and time-consuming, bioelectric measurements in difficult-to-access primary tissues.
The authors have nothing to disclose.
The authors would like to thank Christopher Fladd and SPARC BioCentre at the Hospital for Sick Children for their help in conducting the membrane potential assays measuring CFTR and ENaC. This work was supported by the CFIT Program with funding provided by CF Canada and the Sick Kids Foundation. This work was funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-148). This study was funded by the Government of Ontario. S.X. was supported by the Canadian Association of Gastroenterology (CAG) Ph.D. Scholarship.
Amiloride | Spectrum Chemical | TCI-A2599-5G | Dissolved in DMSO and stored at -20 °C |
CFTRInh-172 | CF Foundation Therapeutics | Dissolved in DMSO and stored at -20 °C | |
EMEM, 1xs | Wisent | 320-005-CL | |
Fetal Bovine Serum (FBS) | Wisent | 080-450 | |
FLIPR Membrane Potential Dye | Molecular Devices | R8042 | Stored at 4 °C |
Forskolin | Sigma-Aldrich | F3917 | Dissolved in DMSO and stored at -20 °C |
Gluconic acid lactone or (D-(+)-Gluconic acid δ-lactone) | Sigma-Aldrich | G4750 | Stored at room temperature |
HEPES | Bioshop | HEP001.5 | Stored at room temperature |
Human Bronchial Adenocarcinoma Cell Line (Calu-3) | ATCC | HTB-55 | |
Human Epithelial Colorectal Adenocarcinoma Cell Line (Caco-2) | ATCC | HTB-37 | |
N-Methyl-D-glucamine (NMDG | Sigma-Aldrich | M2004 | Stored at room temperature |
Penicillin-Streptomycin Solution | Wisent | 450-200-EL | |
Phosphate-Buffered Saline (PBS) | Wisent | 311-010-CL | |
Potassium Gluconate | Sigma-Aldrich | P1847 | Stored at room temperature |
Sodium Gluconate | Sigma-Aldrich | G9005 | Stored at room temperature |