Presentation on theme: "Introduction Ion channels and transporters are proteins that regulate the movement of ions across biological membranes. Research on these proteins has."— Presentation transcript:
Introduction Ion channels and transporters are proteins that regulate the movement of ions across biological membranes. Research on these proteins has led to the development of drug therapies for arrhythmias, hypertension, and neurological disorders. These proteins are also potential sites for adverse drug reactions. To screen large numbers of drug candidates for efficacy and safety, it is essential to use HTS assays. FLIPR TETRA was configured with prototype 400nm excitation LEDs and emission filters at 440-480 nm and 565-625 nm and was used to develop a 384-well FRET- based HTS assay employing Voltage Sensor Probe dyes, (VSPs). Changes were induced in membrane potential of rat basophilic leukemia cells, RBL-2H3, containing an inward rectifying potassium channel (K ir ). Potassium chloride depolarized the cell membrane and barium chloride inhibited this response. Ratiometric analysis provided background correction and direct comparison of FRET data. In a prototype model, we show that utilization of VSPs with FLIPR TETRA provides a sensitive and efficient cell-based system for measuring changes in membrane potential brought about by ion channels and transporters. Mechanism of the FRET-based VSPs Voltage Sensor Probes are a Fluorescence Resonance Energy Transfer (FRET) based assay technology used for high-throughput ion channel drug discovery. The membrane-bound, coumarin- phospholipid (CC2-DMPE) FRET donor binds only to the exterior of the cell membrane. The FRET acceptor is a mobile, negatively charged, hydrophobic oxonol [either DiSBAC 2 (3) or DiSBAC 4 (3)], which binds to either side of the plasma membrane in response to changes in membrane potential. Resting cells have a relatively negative potential. Therefore, the two probes associate with the cell membrane exterior, resulting in efficient FRET. Exciting the CC2-DMPE donor probe (at ~405 nm) generates a strong red fluorescence signal (at ~580 nm) from the oxonol acceptor probe. When the membrane potential becomes more positive, as occurs with cell depolarization by KCl, the oxonol probe rapidly translocates (on a sub second time scale) to the other membrane face. Thus, each oxonol probe "senses" and responds to voltage changes in the cell. This translocation separates the FRET pair, and exciting the CC2-DMPE donor probe now generates a strong blue fluorescence signal (at ~460 nm) from the CC2-DMPE probe. Results Figure 3. Barium chloride inhibition of inward rectifying potassium channel response in rat basophilic leukemia cells as determined by VSP ratiometric assay dyes. Barium chloride inhibited the K ir channel in rat basophilic leukemia cells at an IC 50 concentration of 270 M. This was calculated from the 4 parameter curve fit program in Graphpad Prism. Patch clamping data showed an IC 50 concentration of 500 uM 2. As a measure of the overall performance of the assay, Z’ was calculated at the IC 80 value and equaled 0.58. Z’ factor is a tool for the comparison and evaluation of the quality of assays useful for assay optimization. 3 A Z’ factor > 0.5 is considered a valid assay. An example of an assay data screen can be seen in figure 4. Conclusions A prototype configuration of 400 nm excitation LEDs and emission filters at 440-480 nm and 565-625 nm were used in FLIPR TETRA to effectively demonstrate ratiometric Voltage Sensor Probe assays. Ratiometric dye assays and FLIPR TETRA, along with other membrane potential reagents are important tools for ion channel target assay development. A variety of user exchanged LED modules and filter combinations together with ratiometric dyes and simultaneous read 96-, 384- and 1536-well plate formats provides flexibility in measuring changes in membrane potential brought about by ion channels and transporters. Figure 4. FLIPR TETRA Screenworks data traces at 460 nm grouped by concentration. Lt. Green= Buffer Control Blue = Positive Control Apricot on RH side = No cells control Calculation of Ratiometric Data Bias subtracted based on Sample 1 E 460 nm = Red Color E 580 nm = Blue Color Figure 2. Dual wavelength VSP Trace from FLIPR TETRA Ratiometric data analysis provides background correction and a direct comparison of FRET assay data at both 460 nm and 580 nm for each well. Data for the same cells can be compared in both polarized and non polarized states. An example of data from FLIPR TETRA is seen in Figure 2 above. A normalized ratio (Rf/Ro) reflecting background correction and RFU after addition of depolarizing agent divided by the initial RFU is calculated for each well. FLIPR TETRA is set to take readings at both 460 nm and 580 nm before and after addition of KCl buffer (depolarizing compound). Within identical data windows, an average of readings prior to addition and 40-60 seconds post addition are determined. Average readings from wells without cells are subtracted to calculate the baseline corrected values (BLC). Emission ratios for depolarized and polarized states: Emission Ratio Polarized = BLC 460 initial /BLC 580 initial Emission Ratio Depolarized = BLC 460 final /BLC 580 final The response ratio is calculated as: R f /R o = Emission Ratio Depolarized /Emission Ratio Polarized Figure 1. Schematic of VSP FRET-based dye indicating change in membrane potential (Courtesy of Invitrogen) Ratiometric FRET-based Ion Channel Modulation Assay Development on FLIPR TETRA ™ with Voltage Sensor Probes Carole Crittenden, Jennifer McKie, and Joe Jackson (Molecular Devices Corporation, Sunnyvale, CA) and Randall L. Hoffman (Invitrogen Corporation, Madison, WI) Materials VSP Solution 1 160 mM NaCl (Cat# S 5886), 4.5 mM KCl (Cat# P 5405 ), 2 mM CaCl 2 (Cat# C 7902), 1 mM MgCl 2 (Cat# M 4880), 10 mM glucose (Cat# G7021) All from Sigma, St. Louis, MO, 10 mM HEPES (Cat# 15630-080, Invitrogen, Carlsbad, CA), pH 7.4 High K + solution 164 mM KCl (Cat# P 5405 ), 2 mM CaCl 2 (Cat# C 7902), 1 mM MgCl 2 (Cat# M 4880), 10 mM glucose (Cat# G7021) All from Sigma, St. Louis, MO, 10 mM HEPES (Cat# 15630-080, Invitrogen, Carlsbad, CA), pH 7.4 DMSO (Cat# 47,230-1 Aldrich, St. Louis, MO) Barium Chloride (Cat# B 0750, Sigma, St. Louis, MO) Voltage Sensor Probes Set: DisBAC 2 (3) and CC2-DMPE (Cat# K1016), VABSC-1 Background Suppression Dye (Cat# K1019 all from Invitrogen, Carlsbad, CA) RBL-2H3 Rat Basophilic Leukemia Cells (Cat# CRL-2256, ATCC, Manassas, VA) Cell Culture Reagents Eagle’s Minimum Essential Media (Cat# 30-2003, ATCC, Rockville, MD), 10% FBS (Cat# SH-30071, Hyclone, Logan, UT), Trypsin/EDTA (.25% Trypsin/1mM EDTA) (Cat# 25200- 072), Dulbecco’s PBS (Cat# 14040-133, both from Gibco, Carlsbad, CA) Methods Cell Culture RBL-2H3 cells were trypsinized, washed, and plated at 12,500 cells/well in a BD BioCoat™ Poly-d-Lysine coated 384- well plate (Cat# 356663, BD Biosciences, Franklin Lakes, NJ) 18 to 24 hours prior to assay. A no cell control was placed in Column 24 A-F. Preparation of VSP loading buffers CC2-DMPE: A 5 mM stock solution of was prepared in DMSO and stored at –20 o C. On the day of the assay, a working solution was prepared. An equal volume of 10% Pluronic acid was mixed with CC2-DMPE and subsequently diluted to 5 M in VSP-1 solution. The buffer was vigorously mixed and protected from light prior to use. DisBAC 2 (3): A 12 mM stock solution was prepared in DMSO and stored at –20 o C. A 200 mM stock solution of VABSC-1 was prepared in water and stored at RT. On the day of the assay, a 4 M working solution of DisBAC 2 (3) was prepared including 0.21 M VABSC-1 quencher in VSP-1 solution. No special considerations were necessary to utilize the VSP dyes on FLIPR TETRA. Loading Cells Media was removed from all wells of the 384 well plate and replaced with 50 l VSP-1 solution. VSP-1 was removed immediately and replaced with 25 l CC2-DMPE loading buffer and incubated at room temperature for 30 minutes, covered and protected from light. CC2-DMPE loading buffer was removed after 30 minutes and wells were washed with 50l VSP-1 solution. VSP-1 was removed immediately and replaced with 4 M DisBAC 2 (3) and 0.21 M VABSC-1 loading buffer and incubated for 30 minutes at room temperature protected from light. A series of of BaCl 2 (inhibitor) concentrations was added to respective wells at the start of the incubation period. On FLIPR TETRA, readings at both 460 nm and 580 nm were taken for 10 seconds before adding 25 l High K+ solution to the wells and for about 40 seconds after addition. These form the basis of the Ratio. Instrument parameters are listed in Table 1. FLIPR TETRA Instrument Setup Parameters Table 1. Ratiometric setup parameters for FLIPR TETRA ParameterFLIPR TETRA Setting Read Mode 1 Excitation400 nm Read Mode 1 Emission440-480 nm Read Mode 2 Excitation400 nm Read Mode 2 Emission545-625 nm LED Intensity100% Camera Gain20 Camera Exposure0.25 sec each mode Read Interval1 sec for both modes Pipetting Volume 25 l Dispense Height 30 l Dispense Speed 20 l/sec Tip Up Speed10 mm/sec Tips In Well During ReadNo References 1.M. Falconer et.al. High-Throughput Screening for Ion Channel Modulators, Journal of Biomolecular Screening, Volume 7, (5)460-465, (2002) 2.J. Gonzalez, et al. Cell-based assays and instrumentation for screening ion- channel targets, Drug Discovery Today, 4(9): 431-439, (1999) 3.J. Zhang, et. al. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, Journal of Biomolecular Screening, Vol. 4, (2)60-73, (1999)
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