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.

1. Cell plating and differentiation
<ol>
	<li>Culture Calu-3 and Caco-2 cells in EMEM containing 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin (pen/strep) in a T-75 flask.</li>
	<li>Once the cells reach 80-100% confluency, aspirate the medium from the T-75 flask.</li>
	<li>Gently wash the cells once with 10 mL of phosphate-buffered saline (PBS).</li>
	<li>Add 2 mL of prewarmed 0.25% Trypsin/0.1% EDTA to the cell monolayer and place the T-75 flask at 37 &#176;C for approximately 5 min to allow the cells to lift from the culturing flask surface and dissociate into a single-cell suspension.</li>
	<li>Once the cells are lifted from the flask surface, neutralize the reaction with 8 mL of culture medium (step 1.1) for a total of 10 mL of cell suspension. 
	NOTE: Mechanically dissociate cell clumps into a single-cell suspension with a 10 mL serological pipette.</li>
	<li>Plate the cells onto a 96-well plate (~140,000-150,000 cells/well) or a 384-well plate (~45,000-50,000 cells/well) at 30-40% confluency.
	<ol>
		<li>To plate one full 96-well plate, add 1.5 mL of the cell suspension to 18.5 mL of the culture medium in a 50 mL conical tube. After mixing, add 200 &#181;L of the cell suspension to each well.</li>
		<li>To plate one full 384-well plate, add 1 mL of the cell suspension to 17 mL of the culture medium in a 50 mL conical tube. After mixing, add 50 &#181;L of the cell suspension to each well. 
		NOTE: Ensure there are no clumps of cells, and single cells are distributed evenly across each well.</li>
	</ol>
	</li>
	<li>Change the medium every 2 days after plating. If the cells do not reach confluency at the same time between different wells, discard the plate and repeat step 1.5.</li>
	<li>Once seeded, wait for the cells to reach 100% confluency within 3-5 days. Change the medium 24 h prior to the functional studies.</li>
</ol>
2. CFTR functional assay buffer solution preparation
<ol>
	<li>Prepare the sodium-free, chloride-free buffer solution with the reagents listed in Table 1.</li>
	<li>Add the reagents to tissue culture grade, double-distilled H2O (ddH2O). Allow the reagents to dissolve (in a closed container to avoid evaporation) by stirring overnight at room temperature.</li>
	<li>Once the solution is stable, measure the pH. If the pH of the solution exceeds 7.4, add 1 M N-methyl-D-glucamine (NMDG) solution dropwise to adjust the pH to 7.4. 
	NOTE: Do not adjust the pH of the solution with HCl because the solution must remain chloride-free.</li>
	<li>Allow the solution to mix for another 30 min and measure the osmolarity of the solution.
	<ol>
		<li>Reduce the osmolarity of buffer solution to a physiological range of 300 &#177; 5 mOsm/kg.</li>
	</ol>
	</li>
	<li>Filter and store the buffer solution in sterile bottles. 
	&#8203;NOTE: The solution can be stored at 4 &#176;C for up to 6 months. If stored for longer than 6 months, check the pH and osmolarity at room temperature before use.</li>
</ol>
3. CFTR functional assay
<ol>
	<li>Dissolve the membrane potential dye in the sodium-free, chloride-free buffer solution (0.5 mg of dye/1 mL of buffer solution) and warm it to 37 &#176;C. 
	NOTE: Avoid exposing the membrane potential dye powder to light.</li>
	<li>Remove the culture medium from Calu-3 or Caco-2 cell monolayers and add the dye-containing solution to each well (195 &#181;L per well for a 96-well plate, 95 &#181;L per well for a 384-well plate). 
	NOTE: Up to 112 &#181;L can be added per well of 384-well plates.</li>
	<li>Allow the cells to load the dye for 35 min at 37 &#176;C and 5% CO2.</li>
	<li>Move the cells, loaded with dye solution, to the fluorescence plate reader. Take fluorescence measurements from the bottom of the plate, with excitation = 530 &#177; 5 nm, emission = 560 &#177; 5 nm</li>
	<li>Begin by taking continuous baseline readings for 5 min at 30 s intervals.</li>
	<li>Add 5 &#181;L of a low-concentration solution of forskolin or DMSO to each well for a final concentration of 1 &#181;M forskolin (1x). Take a stimulation reading for 20 min with measurements at 15 s intervals.
	<ol>
		<li>For the 96-well plate format, prepare the low-concentration (40 &#181;M forskolin (1:25 dilution)) solution of forskolin by diluting 2 &#181;L of 1 mM forskolin (1,000x) stock solution or DMSO in 48 &#181;L of sodium-free, chloride-free buffer solution. 
		NOTE: The low-concentration 40 &#181;M forskolin is followed by a second dilution (1:40 dilution) in the next step for a final concentration of 1 &#181;M forskolin.</li>
		<li>For the 384-well plate format, prepare the low-concentration (20 &#181;M forskolin (1:50 dilution)) solution by diluting 1 &#181;L of 1 mM (1,000x) forskolin stock solution or DMSO in 49 &#181;L of sodium-free, chloride-free buffer solution. 
		NOTE: The low-concentration 20 &#181;M forskolin is followed by a second dilution (1:20 dilution) in the next step for a final concentration of 1 &#181;M forskolin.</li>
	</ol>
	</li>
	<li>Add 5 &#181;L of low-concentration solution of CFTRInh172 for a final concentration of 10 &#181;M CFTRInh172. Take an inhibition reading for 15 min with measurements at 30 s intervals.
	<ol>
		<li>For the 96-well plate format, prepare the CFTRInh172 low-concentration (400 &#181;M CFTRInh172 (1:25 dilution)) solution by diluting 2 &#181;L of a 10 mM CFTRInh172 (1,000x) stock solution in 48 &#181;L of sodium-free, chloride-free buffer solution. 
		NOTE: The low-concentration 400 &#181;M CFTRInh172 is followed by a second dilution (1:40 dilution) in the next step for a final concentration of10 &#181;M CFTRInh172.</li>
		<li>For the 384-well plate format, prepare the CFTRInh172 low-concentration (200 &#181;M CFTRInh172 (1:50 dilution)) solution by diluting 1 &#181;L of 10 mM CFTRInh172 (1,000x) stock solution in 49 &#181;L of sodium-free, chloride-free buffer solution. 
		&#8203;NOTE: The low-concentration 200 &#181;M CFTRInh172 is followed by a second dilution (1:20 dilution) in the next step for a final concentration of10 &#181;M CFTRInh172.</li>
	</ol>
	</li>
	<li>Quantify the fluorescence intensity measurements of each well, and export the values as a spreadsheet in column format containing individual wells. To calculate forskolin-induced changes, divide the RFU (Relative Fluorescence Units) measurements from each well of the 96- or 384-well plate by the last fluorescence intensity measurement of the baseline reading and plot them.
	<ol>
		<li>Alternatively, quantify CFTRInh172-mediated inhibition response to better confirm the specificity of CFTR function12, as acute treatment with forskolin may result in membrane depolarization through the activity of other chloride channels, such as SLC26A912.</li>
	</ol>
	</li>
	<li>Measure the peak responses as the maximum fluorescence intensity measurement from baseline during forskolin stimulation. Use this measurement or area under the curve to quantify the CFTR response.</li>
</ol>
4. ENaC functional assay buffer solution preparation
<ol>
	<li>Prepare the high-sodium, chloride-free buffer solution with the reagents listed in Table 2.</li>
	<li>Once the solution is stable, measure the pH. If the pH of the solution is below 7.4, add 1 N NaOH solution dropwise to adjust the pH to 7.4. 
	NOTE: Do not adjust the pH of the solution with HCl because the solution must remain chloride-free.</li>
	<li>Reduce the osmolarity of buffer solution to a physiological range of 300 &#177; 5 mOsm/kg.</li>
	<li>Filter and store the buffer solution in sterile bottles at 4 &#176;C for up to 6 months. 
	&#8203;NOTE: If storage is longer than 6 months, check the pH and osmolarity at room temperature before use.</li>
</ol>
5. ENaC functional assay
<ol>
	<li>Dissolve the membrane potential dye in the high-sodium, chloride-free buffer solution (0.5 mg of dye/1 mL of buffer solution) and warm it to 37 &#176;C. 
	NOTE: Avoid exposing the membrane potential dye powder to light.</li>
	<li>Remove the culture medium from Calu-3 or Caco-2 cell monolayers and add the dye-containing solution to each well (195 &#181;L per well for 96-well plates, 95 &#181;L per well for 384-well plates). 
	NOTE: Avoid exposing the dye solution to light.</li>
	<li>Allow the cells to load dye for 35 min at 37 &#176;C and 5% CO2.</li>
	<li>Move the cells, loaded with dye solution, to the fluorescence plate reader. Read fluorescence measurements from the bottom of the plate, with excitation = 530 &#177; 5 nm, emission = 560 &#177; 5 nm.</li>
	<li>Begin by taking continuous baseline readings for 5 min at 30 s intervals.</li>
	<li>To inhibit ENaC function, add 5 &#181;L of low-concentration solution amiloride or low-concentration solution DMSO dissolved in the high sodium, zero chloride buffer solution for a final concentration of 10 &#181;M amiloride.
	<ol>
		<li>For the 96-well plate format, prepare the low-concentration (400 &#181;M amiloride (1:25 dilution)) solution of amiloride by diluting 2 &#181;L of 10 mM amiloride (1,000x) stock solution in 48 &#181;L of high-sodium, chloride-free buffer solution. 
		NOTE: The low-concentration 400 &#181;M amiloride is followed by a second dilution (1:40 dilution) in the next step for a final concentration of 10 &#181;M amiloride.</li>
		<li>For the 384-well plate format, prepare the low-concentration (200 &#181;M amiloride (1:50 dilution)) solution of amiloride by adding 1 &#181;L of 10 mM amiloride (1,000x) stock solution in 49 &#181;L of high-sodium, chloride-free buffer solution. 
		NOTE: The low-concentration 200 &#181;M amiloride is followed by a second dilution (1:20 dilution) in the next step for a final concentration of 10 &#181;M amiloride.</li>
	</ol>
	</li>
	<li>Take an inhibition reading for 60 min with measurements at 45 s intervals.</li>
	<li>Repeat step 3.7 for data analysis.</li>
	<li>Measure the amiloride response as the percent inhibition from baseline to the point of plateau, approximately 15-20 min after amiloride addition. Use this difference to quantify the ENaC response.</li>
</ol>

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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+platform+for+high-throughput+therapy+testing+on+iPSC-derived+lung+progenitor+cells+from+cystic+fibrosis+patients.">A new platform for high-throughput therapy testing on iPSC-derived lung progenitor cells from cystic fibrosis patients.</a> Stem Cell Reports. 16 (11), 2825-2837 (2021).</li><li>Hu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Embryonic+Kidney+293+Cells:+a+vehicle+for+biopharmaceutical+manufacturing,+structural+biology,+and+electrophysiology.">Human Embryonic Kidney 293 Cells: a vehicle for biopharmaceutical manufacturing, structural biology, and electrophysiology.</a> Cells, Tissues, Organs. 205 (1), 1-8 (2018).</li><li>Hadida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide+(VX-770,+ivacaftor),+a+potent+and+orally+bioavailable+CFTR+potentiator.">Discovery of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, ivacaftor), a potent and orally bioavailable CFTR potentiator.</a> Journal of Medicinal Chemistry. 57 (23), 9776-9795 (2014).</li><li>Chen, M. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+optimization+of+novel+high-throughput+assays+for+human+epithelial+sodium+channels.">Validation and optimization of novel high-throughput assays for human epithelial sodium channels.</a> Journal of Biomolecular Screening. 20 (2), 242-253 (2015).</li><li>Maitra, R., Sivashanmugam, P., Warner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+membrane+potential+assay+to+monitor+CFTR+function+and+inhibition.">A rapid membrane potential assay to monitor CFTR function and inhibition.</a> Journal of Biomolecular Screening. 18 (9), 1132-1137 (2013).</li><li>Mall, M. 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Lung Cellular and Molecular Physiology. 312 (6), 912-925 (2017).</li><li>Frelin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amiloride+and+its+analogs+as+tools+to+inhibit+Na++transport+via+the+Na++channel+the+Na+/H++antiport+and+the+Na+/Ca2++exchanger.">Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel the Na+/H+ antiport and the Na+/Ca2+ exchanger.</a> Biochimie. 70 (9), 1285-1290 (1988).</li><li>Di Paola, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLC6A14+is+a+genetic+modifier+of+cystic+fibrosis+that+regulates+Pseudomonas+aeruginosa+attachment+to+human+bronchial+epithelial+cells.">SLC6A14 is a genetic modifier of cystic fibrosis that regulates Pseudomonas aeruginosa attachment to human bronchial epithelial cells.</a> mBio. 8 (6), 02073 (2017).</li><li>Schiavi, S. 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The authors declare no competing interests.

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 &#181;M 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.

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 &#34;hits&#34; 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.

<table><tbody><tr><td>Amiloride</td><td>Spectrum Chemical</td><td>TCI-A2599-5G</td><td>Dissolved in DMSO and stored at -20 &amp;deg;C</td></tr><tr><td>CFTRInh-172</td><td>CF Foundation Therapeutics</td><td></td><td>Dissolved in DMSO and stored at -20 &amp;deg;C</td></tr><tr><td>EMEM, 1xs</td><td>Wisent</td><td>320-005-CL</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Wisent</td><td>080-450</td><td></td></tr><tr><td>FLIPR Membrane Potential Dye</td><td>Molecular Devices</td><td>R8042</td><td>Stored at 4 &amp;deg;C</td></tr><tr><td>Forskolin</td><td>Sigma-Aldrich</td><td>F3917</td><td>Dissolved in DMSO and stored at -20 &amp;deg;C</td></tr><tr><td>Gluconic acid lactone or (D-(+)-Gluconic acid &amp;delta;-lactone)</td><td>Sigma-Aldrich</td><td>G4750</td><td>Stored at room temperature</td></tr><tr><td>HEPES</td><td>Bioshop</td><td>HEP001.5</td><td>Stored at room temperature</td></tr><tr><td>Human Bronchial Adenocarcinoma Cell Line (Calu-3)</td><td>ATCC</td><td>HTB-55</td><td></td></tr><tr><td>Human Epithelial Colorectal Adenocarcinoma Cell Line (Caco-2)</td><td>ATCC</td><td>HTB-37</td><td></td></tr><tr><td>N-Methyl-D-glucamine (NMDG</td><td>Sigma-Aldrich</td><td>M2004</td><td>Stored at room temperature</td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Wisent</td><td>450-200-EL</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Wisent</td><td>311-010-CL</td><td></td></tr><tr><td>Potassium Gluconate</td><td>Sigma-Aldrich</td><td>P1847</td><td>Stored at room temperature</td></tr><tr><td>Sodium Gluconate</td><td>Sigma-Aldrich</td><td>G9005</td><td>Stored at room temperature</td></tr></tbody></table>

a fluorescence-based assay of membrane potential for high-throughput functional study of two endogenous ion channels in two 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.

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 &#177; 3 standard deviations from the mean forskolin stimulation (black points) or DMSO control (grey points), which corresponded to a Z&#39; 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 &#181;M) (Figure 2A) and high concentrations (50 &#181;M) of amiloride (Figure 2A,B) and in Calu-3 cells with a high concentration of amiloride (50 &#181;M) (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 &#177; 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&#39; factor of 0.629 with acute treatment using amiloride (50 &#181;M) (Figure 2C), which indicates that these parameters support high-throughput screening.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63528/63528fig01.jpg" /> 
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 &#181;M)and DMSO control in Caco-2 cells. (B) Box and whisker plot of forskolin stimulation in Caco-2 and Calu-3 cells (**** P &#60; 0.0001, n &#62; 3 biological replicates, n &#62; 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; &#916;F/F0 = change in fluorescence. <a href="https://www.jove.com/files/ftp_upload/63528/63528fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63528/63528fig02.jpg" /> 
Figure 2: ENaC inhibition measurements through membrane hyperpolarization in Caco-2 and Calu-3 cells. (A) Representative trace of ENaC inhibition with amiloride (10 &#181;M)relative to DMSO control in Caco-2 cells. (B) Box and whisker plot of amiloride inhibition in Caco-2 and Calu-3 cells (**** P &#60; 0.0001, n &#62; 3 biological replicates, n &#62; 3 technical replicates). (C)&#160;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; &#916;F/F0 = change in fluorescence. <a href="https://www.jove.com/files/ftp_upload/63528/63528fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>NMDG (N-Methyl-D-glucamine)</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td>Gluconic Acid Lactone</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td>Potassium Gluconate</td>
			<td>3 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>
Table 1: Reagents and their corresponding concentrations for CFTR functional assay with zero sodium and chloride. Abbreviation:&#160;CFTR = Cystic Fibrosis Transmembrane Conductance Regulator.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>Sodium Gluconate</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td>Potassium Gluconate</td>
			<td>3 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>
Table 2: Reagents and their corresponding concentrations for ENaC functional assay buffer with high sodium, zero chloride.&#160;Abbreviation: ENaC = Epithelial sodium (Na) Channel.

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A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

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 ...

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Research

JoVE Journal

Biology

2.0K Views.  Hospital for Sick Children. 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. 

Video: A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Biochemistry

Pharmacological Targeting of Ion Channels to Treat Solid Tumors

Screening Ion Channels in Cancer Cells

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.

Author Spotlight: Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches 

The pharmacological targeting of ion channels is a promising approach to treating solid tumors. Detailed protocols are provided for characterizing ion channel function in cancer cells and assaying the effects of ion channel modulators on cancer viability.

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development ...

Cancer Research

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Neuroscience

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Evaluating Electroporation Effects on Genetically Engineered Spiking HEK Cells

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could&#160;be efficiently evaluated.

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be ...

Bioengineering

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...