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 Sl...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="723">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">DMEM, High Glucose</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Leibovitz&#39;s L-15 Medium</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">11415-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Penicillin-Streptomycin (10,000 U/ml)</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Geneticin G-418 Sulfate</td>
			<td style="width:172px;">American Bioanalytical</td>
			<td style="width:119px;">AB05057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">HBSS</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Fibronectin from human plasma</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:119px;">F0895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Albumin from chicken egg white</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">DPBS</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Hausser Bright-Line Phase Hemocytometer</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Carbamoylcholine chloride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">SFLLR-NH2 trifluoroacetate salt</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Finnpipette (5-50 &#181;l)</td>
			<td style="width:172px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">4662090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">BIND Scanner System</td>
			<td style="width:172px;">X-BODY Biosciences</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">384-well TiO2 coated plates</td>
			<td style="width:172px;">X-BODY Biosciences</td>
			<td style="width:119px;">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

Research

JoVE Journal

Bioengineering

10.2K vues.  Yale School of Medicine.  Techniques de biocapteurs optiques peuvent détecter des changements dans la masse à proximité de la membrane plasmique dans les cellules vivantes et permettre un suivi de la réponse cellulaire dans les deux cellules et les populations de cellules individuelles. Ce protocole décrit la détection de la modulation des canaux potassiques par G récepteurs couplés aux protéines dans des cellules intactes en utilisant cette approche. 