The authors are grateful to Dr. Yangyang Yan in the laboratory of Dr. Fred Sigworth at Yale University for the generous donation of HEK293 cells&#160;stably expressing rat Slack-B protein. Additionally the authors are grateful to Dr. Sigworth for cryo-electron microscopy homology model of Slack displayed in the video introduction.&#160;This research was supported by NIH Grants DH067517 and NS073943 to L.K.K.

1. Cell Culture (Adapted from Fleming and Kaczmarek18)
<ol>
	<li>Culture cells in appropriate media for greater than 2 but less than 10 passages before use in this assay. Untransfected HEK-293 cells and HEK-293 cells stably expressing rat Slack-B protein are grown in one half Dulbecco&#39;s Modified Eagle medium and one half Leibovitz&#39;s L-15 Medium supplemented with 10% heat inactivated fetal bovine serum and antibiotics. 500 &#181;g/ml&#160;Geneticin (G418) is added to HEK-293 cells stably expressing Slack-B to select for expression of the Slack channel. Passage cells at 70-90% confluence by dissociation from dishes with a 0.25% trypsin-EDTA solution.</li>
</ol>
2. Extracellular Matrix (ECM) Coating of the TiO2 384-well Optical Biosensor Plate with Fibronectin and Ovalbumin
<ol>
	<li>Preparation of the fibronectin ECM mixture and ovalbumin blocking solution
	<ol>
		<li>Prepare a working solution of fibronectin in phosphate buffered saline (PBS) with a final concentration of 5 &#181;g/ml&#160;of fibronectin in 20.0 ml&#160;PBS. From a 1.0 mg/ml&#160;stock concentration, add 100 &#181;l&#160;to 20.0 ml&#160;PBS for a 200-fold dilution. The working solution of fibronectin can be aliquoted and stored at -20 &#176;C after preparation.</li>
		<li>Prepare a 5% stock solution of heat-inactivated ovalbumin in sterile water. Dilute ovalbumin in sterile tissue culture grade water for a 5% solution and heat-inactivate for 30 min at 60 &#176;C. Cool the solution to room temperature and sterile filter the solution. Aliquot the 5% ovalbumin solution and store at 4 &#176;C.</li>
		<li>Prepare a 2% working solution of ovalbumin by diluting the 5% stock solution into PBS. For example, add 12.0 ml of 5% ovalbumin stock solution to 18.0 ml of PBS for a final volume of 30.0 ml.</li>
	</ol>
	</li>
	<li>384-well optical biosensor plate coating
	<ol>
		<li>Hydrate the TiO2 plate with 20.0 &#181;l/well of sterile tissue culture grade water for 15 min. The following steps should be performed with a 16-channel pipettor.</li>
		<li>Wash plate 1x with 50.0 &#181;l PBS per well.</li>
		<li>Add 20.0 &#181;l of the fibronectin working solution to each well and incubate for 2 hr at room temperature to create the ECM.</li>
		<li>Wash plate 1x with 50.0 &#181;l PBS per well.</li>
		<li>Add 40.0 &#181;l of the ovalbumin working solution to each well and incubate overnight at 4 &#176;C to block the plate.</li>
		<li>Wash plate 3x with 50.0 &#181;l PBS per well. ECM coated plates can be stored at 4 &#176;C for up to 48 hr.</li>
	</ol>
	</li>
</ol>
3. Seeding of Cells onto the 384-well Optical Biosensor Plate
<ol>
	<li>Remove growth media from the cells and add trypsin to dislodge the cells from the plate. Collect the cells and centrifuge at 1,000 x g for 1 min. Remove the trypsin media and resuspend cells in growth media.</li>
	<li>Using a hemocytometer or an automated cell counter, determine the concentration of the cells.</li>
	<li>To minimize the effects of evaporation during incubation, 50.0 &#181;l of growth medium per well will be used. Cells should be diluted to achieve the desired number of cells per well in a volume of 50.0 &#181;l of growth medium. For these experiments, 1,000 cells/well were plated onto the biosensor.</li>
	<li>Allow the seeded cells to incubate overnight in growth medium at 37 &#176;C under air/5% CO2.</li>
</ol>
4. Preparation of the Biosensor and Compound Plates for the RWG Optical Biosensor Assay
<ol>
	<li>Remove the plate from the incubator and remove growth media from the cells. Wash the cells 1x with Hanks Balanced Salt Solution (HBSS). Add 25.0 &#181;l of HBSS to each well.</li>
	<li>Place the biosensor plate on the BIND Scanner and allow the cells to equilibrate to room temperature for 1-2 hr.</li>
	<li>Prepare a separate compound plate from which ligands of interest will be added to the biosensor plate during the assay. For these assays, 25.0 &#181;l of solution containing compound was added to each well on the biosensor plate for a total volume of 50.0 &#181;l. As such, compounds in solution were prepared in 2x the desired final concentration.</li>
</ol>
5. Perform the RWG Optical Biosensor Assay
<ol>
	<li>Take a baseline measurement with the Scanner system for wells A1-P12 (half of the SBS 384-well plate) using a maximum resolution of 3.75 &#181;m/pixel for the central 1.5 mm of each well using the BIND Scan software . This baseline scan will take approximately 30 min.</li>
	<li>Add 25.0 &#181;l of the compound solutions to each well for wells A1-P12.</li>
	<li>Take a baseline measurement of wells A13-P24.</li>
	<li>Add 25.0 &#181;l of the compound solutions to each well for wells A13-P24.</li>
	<li>Take a post&#160;compound addition measurement for wells A1-P12, and repeat for wells A13-P24.</li>
</ol>
6. Analysis of Assay Data with BIND View
<ol>
	<li>Open the BIND assay data for the baseline measurement in Windows, and click the &#34;Grid Editor&#34; button to enable cell selection. The software will calculate metrics of interest defined by the user.</li>
	<li>Open the &#34;Plug-ins&#34;, which include the &#34;Cell Finder&#34; and &#34;Baseliner&#34; function. Set parameters in &#34;Cell Finder&#34; so that cells are distinguishable from the background of the biosensor.</li>
	<li>Export the cell map file which contains the &#34;Cell Finder&#34; data for the baseline measurement.</li>
	<li>Repeat steps 6.1-6.2 for post&#160;compound addition data.</li>
	<li>Open the &#34;Baseliner&#34; plug-in and import the cell map from the baseline measurement. The software will calculate the shift in PWV between the background and post&#160;compound data.</li>
	<li>Select &#34;&#916; Cell Mean&#34; for the output of the mean shift in PWV for cells. The data can then be exported to Microsoft Excel for further analysis.</li>
</ol>

<ol>
	<li>Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion Channels of Excitable Membranes. , (1992).</li><li>Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-conducting+functions+of+voltage-gated+ion+channels.">Non-conducting functions of voltage-gated ion channels.</a> Nat. Rev. Neurosci. 7, 761-771 (2006).</li><li>Levitan, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+protein+complexes+associated+with+neuronal+ion+channels.">Signaling protein complexes associated with neuronal ion channels.</a> Nat. Neurosci. 9, 305-310 (2006).</li><li>Shamah, S. M., Cunningham, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+cell-based+assays+using+photonic+crystal+optical+biosensors.">Label-free cell-based assays using photonic crystal optical biosensors.</a> Analyst. 136, 1090-1102 (2011).</li><li>Cunningham, B. T., Laing, L. M. i. c. r. o. p. l. a. t. e. -. b. a. s. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=label-free+detection+of+biomolecular+interactions:+applications+in+proteomics.">label-free detection of biomolecular interactions: applications in proteomics.</a> Expert Rev. Proteomics. 3, 271-281 (2006).</li><li>Peters, M. F., Vaillancourt, F., Heroux, M., Valiquette, M., Scott, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+label-free+biosensors+for+pharmacological+screening+with+cell-based+functional+assays.">Comparing label-free biosensors for pharmacological screening with cell-based functional assays.</a> Assay Drug Dev. 8, 219-227 (2010).</li><li>Fang, Y., Ferrie, A. M., Fontaine, N. H., Mauro, J., Balakrishnan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resonant+waveguide+grating+biosensor+for+living+cell+sensing.">Resonant waveguide grating biosensor for living cell sensing.</a> Biophys. J. 91, 1925-1940 (2006).</li><li>Cunningham, B. T., 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=Label-free+assays+on+the+BIND+system.">Label-free assays on the BIND system.</a> J. Biomol. Screen. 9, 481-490 (2004).</li><li>Hessel, A. a. O., A, A. <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+Theory+of+Wood's+Anomalies+on+Optical+Gratings.">A New Theory of Wood's Anomalies on Optical Gratings.</a> Appl. Optics. 4, 1275-1297 (1965).</li><li>Cunningham, B. T., Laing, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+application+of+label-free+detection+assays+in+drug+screening.">Advantages and application of label-free detection assays in drug screening.</a> Expert Opin. Drug Discov. 3, 891-901 (2008).</li><li>Lee, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+optical+biosensor:+a+tool+for+G+protein-coupled+receptors+pharmacology+profiling+and+inverse+agonists+identification.">Label-free optical biosensor: a tool for G protein-coupled receptors pharmacology profiling and inverse agonists identification.</a> J. Recept. Signal Transduct. Res. 29, 146-153 (2009).</li><li>Bhattacharjee, A., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=For+K++channels,+Na++is+the+new+Ca2.">For K+ channels, Na+ is the new Ca2.</a> Trends Neurosci. 28, 422-428 (2005).</li><li>Santi, C. 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=Opposite+regulation+of+Slick+and+Slack+K++channels+by+neuromodulators.">Opposite regulation of Slick and Slack K+ channels by neuromodulators.</a> J. Neurosci. 26, 5059-5068 (2006).</li><li>Yang, B., Desai, R., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slack+and+Slick+K(Na)+channels+regulate+the+accuracy+of+timing+of+auditory+neurons.">Slack and Slick K(Na) channels regulate the accuracy of timing of auditory neurons.</a> J. Neurosci. 27, 2617-2627 (2007).</li><li>Brown, M. 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=Fragile+X+mental+retardation+protein+controls+gating+of+the+sodium-activated+potassium+channel.">Fragile X mental retardation protein controls gating of the sodium-activated potassium channel.</a> 13, 819-821 (2010).</li><li>Zhang, 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=Regulation+of+neuronal+excitability+by+interaction+of+fragile+X+mental+retardation+protein+with+slack+potassium+channels.">Regulation of neuronal excitability by interaction of fragile X mental retardation protein with slack potassium channels.</a> J. Neurosci. 32, 15318-15327 (2012).</li><li>Barcia, G., 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=De+novo+gain-of-function+KCNT1+channel+mutations+cause+malignant+migrating+partial+seizures+of+infancy.">De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy.</a> Nat. Genet. 44, 1255-1259 (2012).</li><li>Fleming, M. R., Kaczmarek, L. K. <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+optical+biosensors+to+detect+modulation+of+Slack+potassium+channels+by+G+protein-coupled+receptors.">Use of optical biosensors to detect modulation of Slack potassium channels by G protein-coupled receptors.</a> J. Recept. Signal Transduct. Res. 29, 173-181 (2009).</li><li>Nelson, C. 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=Targeting+of+diacylglycerol+degradation+to+M1+muscarinic+receptors+by+beta-arrestins.">Targeting of diacylglycerol degradation to M1 muscarinic receptors by beta-arrestins.</a> Science. 315, 663-666 (2007).</li><li>Zhang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J. Biomol. Screen. 4, 67-73 (1999).</li><li>Gribbon, 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=Evaluating+real-life+high-throughput+screening+data.">Evaluating real-life high-throughput screening data.</a> J. Biomol. Screen. 10, 99-107 (2005).</li></ol>

Steven M. Shamah is an employee of X-BODY Biosciences.

The Z prime (Z&#39;) factor is a common statistical method to quantify the quality of a high-throughput screening assay20, and because the screening of the GPCR agonists in this assay occurred in a SBS compatible 384-well assay, provides an excellent measure of the robustness and validity of this assay21. A Z&#39; value of 1 indicates a theoretically ideal assay with an infinite number of data points with nonexistent standard deviations. A Z&#39; value of between 0.5-1 is considered an excellent ass...

Examining living cells in their physiologically relevant context is crucial to understanding the biological functions of cellular targets. However, assays examining the interaction between channels and regulatory cytoplasmic proteins, such as coimmunoprecipitation assays, generally provide little information on the time course of interactions in living cells. The majority of current cell based assays measure a specific cellular event, such as the translocation of a fluorescently tagged protein of interest. These assays require modifications of the proteins of interest, which can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target. Noninvasive cell based assays, which do not require manipulation of the system, provide a continuous measurement of cellular activity and allow studies of cells in their most physiologically relevant state4.
Optical biosensors are designed to produce a measureable change in a characteristic of the light that is coupled to the sensor surface. Optical biosensors that utilize evanescent waves, which include surface plasmon resonance (SPR) and resonance wavelength grating (RWG) techniques, have been primarily used to detect the affinities and kinetics of molecules binding to their biological receptors immobilized on the sensor surface. More recently, commercially available RWG biosensors have been developed to allow the detection of the relative change in mass within the bottom portion of cells adherent to the sensor surface at high spatial resolution (up to 3.75 &#181;m/pixel)5. These biosensors can detect changes in mass near the plasma membrane of living cells that result from stimulation, such as cell adhesion and spreading, toxicity, proliferation&#160;and signaling pathways elicited by a variety of cell surface receptors including G-protein coupled receptors (GPCRs)6. RWG biosensors detect the relative change in the index of refraction as a function of the relative change in mass within 150 nm of the biosensor7. When cells are plated to the biosensor, this change in the index of refraction reflects the change in mass near the plasma membrane of the cell resulting from a change in cellular adherence to the biosensor. This protocol will discuss the detection of channel-protein interactions using RWG biosensors.
RWG data acquisition systems consist of several components. Because the X-BODY Biosciences BIND Scanner was used for these experiments, we shall refer to the components for this particular system, which consists of a plate reader, associated biosensors, BIND Scan acquisition software, and BIND View analysis software8. The photonic crystal biosensors, referred to hereafter as optical biosensors, are composed of a periodic arrangement of a dielectric and material in 2 or 3 dimensions which prevents the propagation of light at specific wavelengths and directions. Photonic crystal structures are based on a phenomenon called Wood&#39;s anomaly. First discovered by Wood in 1902, these anomalies are effects observed in the spectrum of light reflected by optical diffraction gratings where rapid variations in the intensity of particular diffraction orders occur in certain narrow frequency bands. In 1962, Hessel and Oliner presented a new theory of Wood&#39;s anomalies in which finite period grating generates a standing resonance wavelength9. In the optical biosensors described here, Wood&#39;s anomalies are used to produce a RWG biosensor that when stimulated with white light reflects only very narrow band of wavelengths (typically between 850-855 nm).
Individual biosensors consist of a low-refractive index plastic material with a periodic surface structure which is coated with a thin layer of the high-refractive dielectric material titanium dioxide (TiO2). When the biosensor is illuminated with a broad wavelength light source, the optical grating of photonic crystals reflects a narrow range of wavelengths of light which is measured by a spectrophotometer in the BIND Scanner. The peak intensity of the reflected wavelengths (Peak Wavelength Value, or PWV) is then calculated from the signal. The PWV of light shifts upon a change in the index of refraction as a result of an increase or decrease in mass within the proximity (~150 nm) of the biosensor surface. Optical biosensors are incorporated into a standard Society for Biomolecular Sciences (SBS) 384-well microplate for the assays used in these experiments.
RWV biosensors are used to detect changes upon addition of exogenous signals in living cells10. Cells are directly cultured onto the surface of a RWG biosensor and the change in the local index of refraction is monitored upon treatment with specific stimuli. The direction of change in mass at the biosensor can be determined, because an increase in the local index of refraction results from an increase in mass near the biosensor&#160;and is measured as an increase in PWV. Conversely a decrease in mass produces a decrease in PWV. The change in detected PWV can result from numerous cellular events, including changes in cell adhesion, protein recruitment/release, endocytosis and recycling, exocytosis, apoptosis, and cytoskeletal rearrangement. For example, the assay can be detect changes in channel-protein interactions between potassium channels and other cytoplasmic or cytoskeletal components. The event, however, must occur within the ~150 nm detection zone near the biosensor, and in the case of an attached cell, near the plasma membrane. Acquisition of cellular responses is performed with BIND Scan software and an image representation of each of the 384 wells in the SBS plate is generated. This system has a resolution of up to 3.75 &#181;m/pixel, allowing detection of events in single cells, and can be used either with cell lines (such as HEK293 or CHO cells) or with more physiologically relevant primary cells. Image analysis with BIND View software allows the detection of cellular responses in both individual and populations of cells. As the biosensors are incorporated into standard SBS 384-well microplates, the system is readily adaptable to high-throughput screening (HTS).
Resonance-wavelength grating optical biosensors have previously been used to detect changes in mass near the plasma membrane of cells following activation of GPCRs11. Our laboratory has cloned two sodium-activated (KNa) potassium channels, Slack-B and Slick12. The activity of both Slack-B and Slick channels has been shown to be very strongly influenced by direct activation of protein kinase C (PKC)13. Activation of G&#945;q protein coupled receptors, such as the M1 muscarinic receptor and the mGluR1 metabotropic glutamate receptor, potently regulates channel activity through PKC activation. These KNa channels contribute to neuronal adaptation during sustained stimulation and regulate the accuracy of timing of action potentials14. Slack channels are known to interact with a variety of cytoplasmic signaling molecules, including FMRP, the Fragile X Mental Retardation protein15,16. Mutations in Slack result in Multiple Migrating Partial Seizures of Infancy (MMPSI), an early onset form of epilepsy which results in severe developmental delay17.

<table><tbody><tr><td>DMEM, High Glucose</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Life Technologies</td><td>11415-064</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Geneticin G-418 Sulfate</td><td>American Bioanalytical</td><td>AB05057</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies</td><td>14025-092</td><td></td></tr><tr><td>Fibronectin from human plasma</td><td>Sigma-Aldrich</td><td>F0895</td><td></td></tr><tr><td>Albumin from chicken egg white</td><td>Sigma-Aldrich</td><td>A5378</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Hausser Bright-Line Phase Hemocytometer</td><td>Fisher Scientific</td><td>02-671-6</td><td></td></tr><tr><td>Carbamoylcholine chloride</td><td>Sigma-Aldrich</td><td>C4382</td><td></td></tr><tr><td>SFLLR-NH2 trifluoroacetate salt</td><td>Sigma-Aldrich</td><td>S8701</td><td></td></tr><tr><td>Finnpipette (5-50 &amp;micro;l)</td><td>Thermo Scientific</td><td>4662090</td><td></td></tr><tr><td>BIND Scanner System</td><td>X-BODY Biosciences</td><td>N/A</td><td></td></tr><tr><td>384-well TiO&lt;sub&gt;2&lt;/sub&gt; coated plates</td><td>X-BODY Biosciences</td><td>TiO-96-CM</td><td></td></tr></tbody></table>

use of label-free optical biosensors to detect modulation of potassium channels by g-protein coupled receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Slack-B stably transfected HEK-293 cells and control untransfected HEK-293 cells were seeded at 1,000&#160;cells/well on 384-well, ECM coated RWG biosensor plates. Images from the central 1.5 mm2 of the biosensor were recorded at a resolution of 3.75 &#181;m/pixel (Figure 1). Density gradient maps of mass were generated pre&#160;and 30 min post&#160;compound addition with the BIND Scan software. The BIND View software was utilized to determine the shift in PWV upon GPCR agonist addition by sub...

Watch this Scientific Journal Video about Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors at JoVE.com

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

use-label-free-optical-biosensors-to-detect-modulation-potassium

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Bioengineering

10.2K Views.  Yale School of Medicine. Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Video: Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Biochemistry

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. ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

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 ...

Biology

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

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, ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Medicine

Functional Site-Directed Fluorometry in Native Skeletal Muscle Cells

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels&#39; activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

Author Spotlight: Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability  

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, ...

Optical Biosensing

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable signal such as fluorescence,&#160;or these sensors can be label-free&#160;and use the changes in optical properties, such as refractive index, to sense for the binding of the target molecule. This video introduces both label and label-free optical biosensors, demonstrates their use in the laboratory, and shows some applications of the technology.

Types of Optical Biosensing; Surface Plasmon Resonance SPR

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable signal such as fluorescence, or these sensors can be label-free&#160;and use the changes in optical properties, such as refractive index, to sense for the binding of the target molecule. This video introduces both label and label-free optical biosensors, demonstrates their use in the laboratory, and shows some applications of the technology.

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable ...