FMP2 - Molecular Devices

An improved FMP dye formulation for the screening of
constitutively active channels
Guy Servant
MDS Analytical Technologies
Drug Discovery Forum
April 25, 2009
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Introduction
~4 years ago, a scientist noticed an atypical effect of channel blockers
in one of our assay using FMP. Results suggested that the dye
formulation caused our target channel to become depolarized during
dye loading
Communicated results to Molecular Devices who decided to work on a
new dye formulation
Senomyx tested several versions over the last few years
Our data suggests that FLIPR ® Membrane Potential Two (FMP2)
improves assay statistics and robustness when working with channels
exhibiting constitutive activity
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Properties of a Constitutively Open Sodium Channel
Open probability is 0.2 for individual channels
Recording at -80 mV
Extracellular
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+
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+ +
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+ +
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+
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Extracellular
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Intracellular
+
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Intracellular
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Limitation of FLIPR Membrane Potential Dye
Assay
Cannot control resting membrane potential
• If cells become depolarized during dye load, inward current will
decrease
Outward Current
Outward Current
Depolarization
Assay
Window
-80 +80 -80 +80
mV
mV
Inward Current
μA
Inward Current
μA
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Cells Loaded with FMP2 are Less Depolarized
• Conditions:
• Channel transiently expressed in HEK cells
• Cells washed off media with No-sodium buffer (NMDG buffer)
• Cells loaded with FLIPR ® Membrane Potential (FMP) or FLIPR ® Membrane
Potential Two (FMP2) in NMDG buffer
• Basal fluorescence acquired
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FMP2 Dye Increases ΔF/F by Almost 2 Fold
• Conditions:
• Channel transiently transfected in HEK cells
• Cells washed off media with No-sodium buffer (NMDG buffer)
• Cells loaded with FMP or FMP2 in NMDG buffer
• Cells stimulated with 1:1 of 140 mM NaCl buffer (NaCl buffer)
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With FMP, Blocker Causes Hyperpolarization
• Conditions:
• Channel transiently transfected in HEK cells
• Cells washed off media with No-sodium buffer (NMDG buffer)
• Cells loaded with FMP in NMDG buffer
• Cells stimulated with 1:1 of 140 mM NaCl buffer (NaCl buffer)
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With FMP2, No Hyperpolarization
• Conditions:
• Channel transiently transfected in HEK cells
• Cells washed off media with No-sodium buffer (NMDG buffer)
• Cells loaded with FMP in NMDG buffer
• Cells stimulated with 1:1 of 140 mM NaCl buffer (NaCl buffer)
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FMP2 Provides Robust Assay Window
Sodium ion channel blocker assay statistics using FMP2 dye.
All experiments were performed on a FLPR 3 instrument.
Experiment
ΔF, mean ± 1 st.
dev. (RFU)
Fmin, mean ± 1 st.
dev. (RFU) ΔF/Fmin Z′ value
1 5589 ± 729 9380 ± 728 0.60 0.89
2 10050 ± 808 6374 ± 659 1.58 0.66
3 11706 ± 555 7999 ± 609 1.46 0.73
Average 1.21 0.76
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Dose-Response Analysis With Blockers
Bottom asymptote is
better defined
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Blockers are ~3 Times More Potent with FMP2-Loaded Cells
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Our Observations with FMP2
When working with channels exhibiting constitutive activity
• 1) Cells loaded with FMP2 are less depolarized
• 2) FMP2 Dye increases ΔF/F by almost 2 Fold and provides robust assay
window
• 3) Use of FMP2 eliminates the hyperpolarization effect of channel blockers
• 4) Channel blocker IC50s are more accurate when obtained from FMP2-
loaded cells
When dealing with any new target, testing performances of both FMP
and FMP2 in parallel is recommended as it is difficult to predict if FMP2
will necessarily be superior
FMP2 is available for MDS AT as a custom kit
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Acknowledgements
Ian Ford Paul Brust Cyril Redcrow
Sumita Ray Ning Hung Vivian Nguy
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