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

Summary

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.

Abstract

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 complex^2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein'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 (K_Na) channels by GPCRs.

Introduction

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's behavior and decrease the physiological relevance of t....

Protocol

1. Cell Culture (Adapted from Fleming and Kaczmarek¹⁸)

1. 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's Modified Eagle medium and one half Leibovitz's L-15 Medium supplemented with 10% heat inactivated fetal bovine serum and antibiotics. 500 µg/ml 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....

Results

Slack-B stably transfected HEK-293 cells and control untransfected HEK-293 cells were seeded at 1,000 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 µm/pixel (Figure 1). Density gradient maps of mass were generated pre and 30 min post compound addition with the BIND Scan software. The BIND View software was utilized to determine the shift in PWV upon GPCR agonist addition by subtracting the PWV pos.......

Discussion

The Z prime (Z') 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' value of 1 indicates a theoretically ideal assay with an infinite number of data points with nonexistent standard deviations. A Z' value of between 0.5-1 is considered an excellent ass.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM, High Glucose	Life Technologies	11965-092
Leibovitz's L-15 Medium	Life Technologies	11415-064
Penicillin-Streptomycin (10,000 U/ml)	Life Technologies	15140-122
Geneticin G-418 Sulfate	American Bioanalytical	AB05057
HBSS	Life Technologies	14025-092
Fibronectin from human plasma	Sigma-Aldrich	F0895
Albumin from chicken egg white	Sigma-Aldrich	A5378
DPBS	Life Technologies	14190-144
Hausser Bright-Line Phase Hemocytometer	Fisher Scientific	02-671-6
Carbamoylcholine chloride	Sigma-Aldrich	C4382
SFLLR-NH2 trifluoroacetate salt	Sigma-Aldrich	S8701
Finnpipette (5-50 µl)	Thermo Scientific	4662090
BIND Scanner System	X-BODY Biosciences	N/A
384-well TiO2 coated plates	X-BODY Biosciences	TiO-96-CM