Name: Author Spotlight: Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches
Uploaded: 2023-06-16T12:00:00
Description: Watch this Scientific Journal Video about Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches at JoVE.com

The authors acknowledge support from the Thomas E. &#38; Pamela M. Mischell Family Foundation to S.S. and the Harold C. Schott Foundation funding of the Harold C. Schott Endowed Chair, UC College of Medicine, to S.S.

1. Immunolabeling ion channels in cultured cells
<ol>
	<li>Preparing the cells and experimental set-up
	<ol>
		<li>Maintain the cells as an actively growing culture in 75 cm2 culture flasks. Passage the cells once until they become 50%-90% confluent, depending on the doubling time of the cell line being used. 
		NOTE: For the present study, D283 cells, a Group 3 medulloblastoma cell line, were used.</li>
		<li>Collect the cells from the culture flask into a centrifuge tube (15 mL or 50 mL), and add 2 mL...

Pharmacological Targeting of Ion Channels to Treat Solid Tumors

<ol>
	<li>Prevarskaya, N., Skryma, R., Shuba, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+in+cancer:+Are+cancer+hallmarks+oncochannelopathies.">Ion channels in cancer: Are cancer hallmarks oncochannelopathies.</a> Physiological Reviews. 98 (2), 559-621 (2018).</li><li>Rao, R., 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=Ligand-gated+neurotransmitter+receptors+as+targets+for+treatment+and+management+of+cancers.">Ligand-gated neurotransmitter receptors as targets for treatment and management of cancers.</a> Frontiers in Physiology. 13, 839437 (2022).</li><li>Mohr, C. 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=Cancer-associated+intermediate+conductance+Ca2+-activated+K++channel+KCa3.1.">Cancer-associated intermediate conductance Ca2+-activated K+ channel KCa3.1.</a> Cancers. 11 (1), 109 (2019).</li><li>Fels, B., Bulk, E., Petho, Z., Schwab, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+TRP+channels+in+the+metastatic+cascade.">The role of TRP channels in the metastatic cascade.</a> Pharmaceuticals. 11 (2), 48 (2018).</li><li>Eil, R., 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=Ionic+immune+suppression+within+the+tumour+microenvironment+limits+T+cell+effector+function.">Ionic immune suppression within the tumour microenvironment limits T cell effector function.</a> Nature. 537 (7621), 539-543 (2016).</li><li>Haustrate, A., Hantute-Ghesquier, A., Prevarskaya, N., Lehen'kyi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+targeting+ion+channels+and+their+therapeutic+potential.">Monoclonal antibodies targeting ion channels and their therapeutic potential.</a> Frontiers in Pharmacology. 10, 606 (2019).</li><li>Kischel, P., 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=Ion+channels:+New+actors+playing+in+chemotherapeutic+resistance.">Ion channels: New actors playing in chemotherapeutic resistance.</a> Cancers. 11 (3), 376 (2019).</li><li>Tuszynski, J., Tilli, T. 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F., Elghajiji, A., Tosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+system+for+electrophysiology+of+cancer+cell+populations:+A+review.">Ultrasensitive system for electrophysiology of cancer cell populations: A review.</a> Bioelectricity. 1 (3), 131-138 (2019).</li><li>Sengupta, 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=&#945;5-GABAA+receptors+negatively+regulate+MYC-amplified+medulloblastoma+growth.">&#945;5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth.</a> Acta Neuropathologica. 127 (4), 593-603 (2014).</li><li>Jonas, O., 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=First+in+vivo+testing+of+compounds+targeting+Group+3+medulloblastomas+using+an+implantable+microdevice+as+a+new+paradigm+for+drug+development.">First in vivo testing of compounds targeting Group 3 medulloblastomas using an implantable microdevice as a new paradigm for drug development.</a> Journal of Biomedical Nanotechnology. 12 (6), 1297-1302 (2016).</li><li>Kallay, L., 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=Modulating+native+GABAA+receptors+in+medulloblastoma+with+positive+allosteric+benzodiazepine-derivatives+induces+cell+death.">Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death.</a> Journal of Neurooncology. 142 (3), 411-422 (2019).</li><li>Pomeranz Krummel, D. A., 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=Melanoma+cell+intrinsic+GABAA+receptor+enhancement+potentiates+radiation+and+immune+checkpoint+response+by+promoting+direct+and+T+cell-mediated+anti-tumor+activity.">Melanoma cell intrinsic GABAA receptor enhancement potentiates radiation and immune checkpoint response by promoting direct and T cell-mediated anti-tumor activity.</a> International Journal of Radiation Oncology, Biology, Physics. 109 (4), P1040-P1053 (2021).</li><li>Bhattacharya, D., 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=Therapeutically+leveraging+GABAA+receptors+in+cancer.">Therapeutically leveraging GABAA receptors in cancer.</a> Experimental Biology and Medicine. 246 (19), 2128-2135 (2021).</li><li>Mazia, D., Schatten, G., Sale, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+cells+to+surfaces+coated+with+polylysine.+Applications+to+electron+microscopy.">Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy.</a> Journal of Cell Biology. 66 (1), 198-200 (1975).</li><li>Wiatrak, B., Kubis-Kubiak, A., Piwowar, A., Barg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PC12+cell+line:+Cell+types,+coating+of+culture+vessels,+differentiation+and+other+culture+conditions.">PC12 cell line: Cell types, coating of culture vessels, differentiation and other culture conditions.</a> Cells. 9 (4), 958 (2020).</li><li>Baker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+in+cytochemistry+and+electron-microscopy.">Fixation in cytochemistry and electron-microscopy.</a> Journal of Histochemistry and Cytochemistry. 6 (5), 303-308 (1958).</li><li>Chung, J. Y., 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=Histomorphological+and+molecular+assessments+of+the+fixation+times+comparing+formalin+and+ethanol-based+fixatives.">Histomorphological and molecular assessments of the fixation times comparing formalin and ethanol-based fixatives.</a> Journal of Histochemistry and Cytochemistry. 66 (2), 121-135 (2018).</li><li>Crowley, L. C., Christensen, M. E., Waterhouse, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+transmembrane+potential+by+TMRE+staining.">Measuring mitochondrial transmembrane potential by TMRE staining.</a> Cold Spring Harbor Protocols. 2016 (12), (2016).</li><li>Cory, A. H., Owen, T. C., Barltrop, J. A., Cory, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+an+aqueous+soluble+tetrazolium/formazan+assay+for+cell+growth+assays+in+culture.">Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture.</a> Cancer Communications. 3 (7), 207-212 (1991).</li><li>Maro, B., Marty, M. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+recording+of+ligand-gated+ion+channels.">Single-channel recording of ligand-gated ion channels.</a> Nature Protocols. 2 (11), 2826-2841 (2007).</li><li>Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J., van Bree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+assay+of+cells+in+vitro.">Clonogenic assay of cells in vitro.</a> Nature Protocols. 1 (5), 2315-2319 (2006).</li><li>Rafehi, H., 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=Clonogenic+assay:+Adherent+cells.">Clonogenic assay: Adherent cells.</a> Journal of Visualized Experiments. (49), 2573 (2011).</li><li>Scudiero, D. A., 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=Evaluation+of+a+soluble+tetrazolium/formazan+assay+for+cell+growth+and+drug+sensitivity+in+culture+using+human+and+other+tumor+cell+lines.">Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.</a> Cancer Research. 48 (17), 4827-4833 (1988).</li><li>Wang, P., Henning, S. M., Heber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+MTT+and+MTS-based+assays+for+measurement+of+antiproliferative+activity+of+green+tea+polyphenols.">Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols.</a> PLoS One. 5, e10202 (2010).</li><li>Berridge, M. V., Tan, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+cellular+reduction+of+3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium+bromide+(MTT):+subcellular+localization,+substrate+dependence,+and+involvement+of+mitochondrial+electron+transport+in+MTT+reduction.">Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.</a> Archives of Biochemistry and Biophysics. 303 (2), 474-482 (1993).</li><li>Plumb, J. A., Milroy, R., Kaye, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+pH+dependence+of+3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium+bromide-formazan+absorption+on+chemosensitivity+determined+by+a+novel+tetrazolium-based+assay.">Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay.</a> Cancer Research. 49 (16), 4435-4440 (1989).</li><li>Chakrabarti, R., Kundu, S., Kumar, S., Chakrabarti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+A+as+an+enzyme+that+catalyzes+the+reduction+of+MTT+to+formazan+by+vitamin+C.">Vitamin A as an enzyme that catalyzes the reduction of MTT to formazan by vitamin C.</a> Journal of Cellular Biochemistry. 80 (1), 133-138 (2000).</li><li>Dong, G. 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Changes in ion channel function alter intracellular signaling cascades, which can impact the overall functioning of a cell. Over the past decade, it has become increasingly clear that ion channels are important to cancer cell growth and metastasis. Importantly, many ion channels are primary targets for approved therapeutics targeting a broad range of disorders24. Investigators have probed whether ion channels could be anti-cancer targets, and the initial results are promising2</s...

Membrane transport proteins are critical for communication between cells, as well as for maintaining cellular homeostasis. Amongst the membrane transport proteins, ion channels serve to drive the growth and development of cells and to maintain the state of cells in challenging and changing environments. Ion channels have also been reported to drive and support the development of solid tumors, both systemically and in the central nervous system (CNS)1,2. For example, KCa3.1 channels are responsible for regulating membrane potential and controlling cell volume, which is important in cell-cycle regulation. Defective KCa3.1 channels have been reported to contribute to the abnormal proliferation of tumor cells3. Further, ion channels may contribute to the metastatic dissemination of cancers. Transient receptor potential (TRP) channels, for example, are involved in Ca2+ and Mg2+ influx; this influx activates several kinases and heat shock proteins that function to regulate the extracellular matrix surrounding a tumor, which is, in turn, important for initiating cancer metastasis4.
Since ion channels can contribute to the development of cancers, they may also be targets for drug-related cancer treatment. For example, resistance to treatment modalities, including chemotherapy and novel immunotherapy, is related to ion channel function dysregulation5,6,7. In addition, ion channels are emerging as important drug targets to impede the growth and development of cancers, with repurposed small molecule (FDA-approved) drugs being examined, as well as biopolymers, including monoclonal antibodies1,2,8,9. While there has been much progress on this front, ion channel cancer drug discovery remains underdeveloped. This is partly due to the unique challenges of studying ion channels in cancer cells. For example, there are technical limitations in setting up electrophysiology assays for slow-acting compounds and temporal differences in channel activation and drug action. Further, the solubility of compounds can also impede progress, as most of the automated electrophysiology systems commonly in use today utilize hydrophobic substrates, which may contribute to artifacts as a result of compound adsorption. In addition, large bioorganic molecular therapeutics such as natural products, peptides, and monoclonal antibodies are technically challenging to screen using conventional electrophysiology assays10. Finally, the bioelectrical properties of cancer cells remain poorly understood11.
Meanwhile, the immunofluorescence staining of ion channels is often challenging. This is due, in part, to the complexity of their structures and their context in the membrane, which impact the ability to both generate and employ antibodies for microscopy studies. It is especially important that the antibodies used to stain ion channels are validated for specificity, affinity, and reproducibility. Commercial antibodies for ion channels should be considered based on their validation strategy and publication record. Experiments should include negative controls to demonstrate the lack of nonspecific binding by either knockdown or knockout of the target protein. Alternatively, cell lines in which the target protein is absent or in low abundance based on mRNA or protein determinations may serve as negative controls. For example, this study shows the localization of the (GABA) receptor&#160;subunit Gabra5 in a medulloblastoma cell line (D283). D283 cells with an siRNA knockdown and Daoy&#160;cells, another cerebellar medulloblastoma cell line, were stained for Gabra5 and showed no appreciatable staining (data not shown).
Here, methods are presented to analyze and assay ion channel function, as well as the effect of ion channel modulators on cancer cells. Protocols are provided for (1) staining cells for an ion channel, (2) testing the polarized state of mitochondria, (3) establishing ion channel function using electrophysiology, and (4) in vitro drug validation. These protocols emphasize studies of the type A gamma-aminobutyric acid (GABAA) receptor2,12,13,14,15,16, a chloride anion channel and major inhibitory neurotransmitter receptor. However, the methods presented here apply to studying many other cancer cells and ion channels.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABS SpectraMax Plate Reader</td>
			<td>Molecular Devices</td>
			<td>ABS</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Invitrogen</td>
			<td>00-4555-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Flor 488</td>
			<td>Invitrogen</td>
			<td>A32723</td>
			<td>Goat Anti-Rabbit</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td>Lacks vitamin A</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>LABCONCO</td>
			<td>302381101</td>
			<td>Class II, Type A2</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1606-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Fisher Scientific</td>
			<td>13-998-211</td>
			<td>Heracell VIOS 160i</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C7902</td>
			<td>Dihydrate</td>
		</tr>
		<tr>
			<td>Cell Culture Dishes, 150 mm</td>
			<td>Fisher Scientific</td>
			<td>12-600-004</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Cell Culture Flasks, 75 cm2</td>
			<td>Fisher Scientific</td>
			<td>430641U</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Cell Culture Plates, 6 well</td>
			<td>Fisher Scientific</td>
			<td>353046</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Cell Culture Plates, 96 well</td>
			<td>Fisher Scientific</td>
			<td>353072</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>EP-5804R</td>
			<td>Refrigerated</td>
		</tr>
		<tr>
			<td>Corning CoolCell</td>
			<td>Fisher Scientific</td>
			<td>07-210-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-553-450</td>
			<td>Corning brand</td>
		</tr>
		<tr>
			<td>D283 Med</td>
			<td>ATCC</td>
			<td>HTB-185</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO Mounting Media</td>
			<td>EMS</td>
			<td>17989-97</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Life Sciences</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>DMEM/F12, base media</td>
			<td>Fisher Scientific</td>
			<td>11330-032</td>
			<td>With phenol red</td>
		</tr>
		<tr>
			<td>DMEM/F12, phenol red free</td>
			<td>Fisher Scientific</td>
			<td>21041-025</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>STEMCELL</td>
			<td>78006.1</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Abcam</td>
			<td>AB120081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor, Basic</td>
			<td>Millipore</td>
			<td>GF003</td>
			<td></td>
		</tr>
		<tr>
			<td>GARBA5 Antibody</td>
			<td>Aviva</td>
			<td>ARP30687_P050</td>
			<td>Rabbit Polyclonal</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Mounting Medium</td>
			<td>EMS</td>
			<td>17989-60</td>
			<td>With DAPI+DABCO</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Millipore Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>STEMCELL</td>
			<td>7980</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>HyClone</td>
			<td>SH3023701</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Open platform</td>
			<td></td>
			<td>With Fiji plugins</td>
		</tr>
		<tr>
			<td>Immuno Mount DAPI</td>
			<td>EMS</td>
			<td>17989-97</td>
			<td></td>
		</tr>
		<tr>
			<td>KRM-II-08</td>
			<td></td>
			<td></td>
			<td>experimental compounds not available from a commercial source</td>
		</tr>
		<tr>
			<td>Leica Application Suite X</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor</td>
			<td>Novus</td>
			<td>N276314100U</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>M9272</td>
			<td>Hexahydrate</td>
		</tr>
		<tr>
			<td>Microscope, Confocal</td>
			<td>Leica</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope, Light</td>
			<td>VWR</td>
			<td>76382-982</td>
			<td>DMiL Inverted</td>
		</tr>
		<tr>
			<td>MTS - Promega One Step</td>
			<td>Promega</td>
			<td>G3581</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipette, 0.5-10 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683914</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipette, 10-100 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683930</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipette, 30-300 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683957</td>
			<td></td>
		</tr>
		<tr>
			<td>Nest-O-Patch</td>
			<td>Heka</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>Gibco</td>
			<td>10888022</td>
			<td>Without vitamin A</td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>Gibco</td>
			<td>12348-017</td>
			<td>Phenol red free</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>NOR-QH-II-66</td>
			<td></td>
			<td></td>
			<td>experimental compounds not available from a commercial source</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>50-998-944</td>
			<td>4 inch width</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>EMS</td>
			<td>RT-15710</td>
			<td></td>
		</tr>
		<tr>
			<td>PATHCHMASTER</td>
			<td>Heka</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion System</td>
			<td>Nanion</td>
			<td>4000120</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>EMS</td>
			<td>RT-15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Bufered Saline</td>
			<td>Fisher Scientific</td>
			<td>AAJ75889K2</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Fisher Scientific</td>
			<td>A3890401</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma Life Sciences</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Port-a-Patch</td>
			<td>Nanion</td>
			<td>21000072</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Life Sciences</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Antibody</td>
			<td>Invitrogen</td>
			<td>MA5-34653</td>
			<td>Rabbit Monoclonal</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fisher Scientific</td>
			<td>NC0758676</td>
			<td>1 mL ampule</td>
		</tr>
		<tr>
			<td>QH-II-66</td>
			<td></td>
			<td></td>
			<td>experimental compounds not available from a commercial source</td>
		</tr>
		<tr>
			<td>Reagent Reservoirs</td>
			<td>VWR</td>
			<td>89094-664</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Slides, 75 x 25 mm</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td>Frosted one side</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Corning</td>
			<td>25-035-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Synth-a-Freeze Medium</td>
			<td>Gibco</td>
			<td>R00550</td>
			<td>Cryopreservation</td>
		</tr>
		<tr>
			<td>TMRE</td>
			<td>Fisher Scientific</td>
			<td>50-196-4741</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>TMRE Kit</td>
			<td>Abcam</td>
			<td>AB113852</td>
			<td>Kit</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>NC0704309</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Gibco</td>
			<td>15-250-061</td>
			<td>Solution, 0.4%</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td>Solution, 0.25%</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>VWR</td>
			<td>97043-562</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Filter Paper</td>
			<td>Fisher Scientific</td>
			<td>09-927-841</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 

Screening Ion Channels in Cancer Cells

The function of ion channels is crucial in cell biology and significantly impacts cancer growth. Our team is focused on targeting ion channels to aid cancer treatments. The field has recently gained a greater understanding of the tumor microenvironment and how cancer cells take hold in healthy tissue, highlighting the importance of ion channels in cancer.One of the biggest challenges in current experiment is the availability of immune-competent animal model that can effectively test therapeutic treatments. We have established that GABA A receptor is a therapeutic vulnerability to disparate cancers and identified a new class of GABA A receptor activators that effectively impair cancer cells. Ion channels play a crucial role in regulating cancer cell behavior, and they hold great potential as target for cancer treatment.Our research has highlighted how an established class of drug can be employed to treat cancer effectively.

Above are select procedures that can be employed to characterize ion channels in cancerous cells. The first protocol highlights the staining of an ion channel. As detailed, there are many challenges when staining an ion channel or, for that matter, any protein that is present in the extracellular membrane. Shown in Figure 1 is the staining for a subunit of the pentameric GABAA receptor. The second protocol highlights the results of testing the polarized state of mitochondria in ca...

Watch this Scientific Journal Video about Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches at JoVE.com

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 ...