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Rikke Schrøder 8 th Ion Channel Retreat 29 th of June, 2010

Biophysical and pharmacological characterization of ligand -gated ion channels on QPatch in multi-hole mode. Rikke Schrøder 8 th Ion Channel Retreat 29 th of June, 2010. Outline. Introduction to QPatch The multi-hole technology Ligand -gated ion channels on QPatch Data

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Rikke Schrøder 8 th Ion Channel Retreat 29 th of June, 2010

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  1. Biophysical and pharmacological characterization of ligand-gated ion channels on QPatch in multi-hole mode Rikke Schrøder 8th Ion Channel Retreat 29th of June, 2010

  2. Outline Introduction to QPatch The multi-hole technology Ligand-gated ion channels on QPatch Data Summary

  3. QPatch introduction • QPatchprovideshighqualitypatchclamp data basedongigaseals • Current is continuouslyrecorded; real-time changesaremeasured • Data analysis is automatic and obtainedwith intuitive and comprehensiveQPatchAssay Software QPatch; a fully automated industrialpatchclamp system

  4. The QPatch family QPatch 8/8X QPatch 16/16X QPatch HT/HTX

  5. QPatch features

  6. QPlate outside Silicon-based patch clamp orifice which replaces conventional glass pipettes Exists in both single-hole and multi-hole versions Exists with either 16 or 48 measurement sites

  7. QPlate inside Upper Flow channel Laminar flow Multiple additions of compounds Glass coated 5µl per liquid addition Waste reservoir 70µl/250µl

  8. QPatch multi-hole technology The chip has multiple (10) holes per well/measurement site The substrate is the silicon chip; gigaseals Flow channelsare the same; laminar flow The measuredcurrent is Itotal Amplifiergainmodifications; measurescurrent up to ± 100nA

  9. Why multi-hole patching? • For assayswith large cell-to-cellheterogeneity in channelexpression • Transientlytransfectedcell lines • Highconsistency in data results • Nearly 100% success rate • Highestpossiblethroughput • Lowest price per data point

  10. B A C Multi-hole challenges B A Is the current rise time the same for single-hole vs. multi-holeexperiments? Is the biophysical/ pharmacologicalprofile the same for single-hole vs. multi-holeexperiments? Is the success rate increased?

  11. Ligand-gated applications on QPatch • Compounds can be applied every 3 seconds (min. time interval) Application protocol for 5 pt antagonist dose-response experiments • Liquidrepetitions (e.g wash) can be applied with intervals of 1 second • Data acquisition and compound application happen simultaneously • Dose-response experiments obtained on the same cell 47 liquid additions á 5 µl per experiment • Intelligent robot logic for handling the pipettes for fast and complex ligand-gated applications

  12. ASIC1a Stability ASIC1a recorded at -60 mV stimulated at pH 6.3 Reference; pH 6.3 Saline; pH 7.3 Minimum cycleduration 180 s gives stable ASIC1a current respons

  13. ASIC1a Rise time ASIC1a 10 – 90% Rise time cursor (pH 6.3) Single-holedata pH 6.8 pH 6.3 ASIC1a 10 – 90% Rise time cursor (pH 6.3) Multi-holedata pH 5.3 Multi-hole Multi-hole Multi-hole Rise time data are the same for Single-holeand multi-hole data

  14. ASIC1a Desensitization Single-mode Multi-mode ASIC1a currentrecorded at pH 6.3 Τavg=2812 ms ± 1783, n=8 ASIC1a currentrecorded at pH 6.3 Τavg=2101 ms ± 398, n=14 ASIC1a desensitizeswithin a fewseconds in bothsingle-hole and multi-hole mode.

  15. ASIC1a Pharmacology ASIC1a currentstimulatedwithincreasingconcentrations of protons (pH 7.3, 6.8, 6.3, 5.3) Resultingconcentrationresponseplot EC50 = 210 nM

  16. ASIC1a IV Single-hole ErevNa (avg) = 67 mV ± 9 mV, n=4 ErevNa (theoretical) = 68 mV

  17. ASIC1a IV Multi-hole ErevNa (avg) = 73 mV ± 4 mV, n=12 ErevNa (theoretical) = 68 mV

  18. GABAA Rise time 10 – 90 % Rise time cursor (12 µM GABA) Single-holedata 10 – 90 % Rise time cursor (12 µM GABA) Multi-holedata Rise time for GABAA is similar for single-hole vs. multi-hole data

  19. GABAA Pharmacology Leaksubtracted GABA α1β2γ2 currentsin responseto increasingconcentrations of GABA (mM) NormalizedGABAresponsesfittedto the Hill equation EC50 = 12 µM

  20. GABAA Success rate Multi-holetechnologyincreases the successrate; from 37% to 93%

  21. GABAA IV Single-hole GABA currentstimulatedwith 10 µM GABA and recorded at Vhold -60, -40, -20, 0, 20, 40 mV

  22. GABAA IV Multi-hole GABA currentstimulatedwith 10 µM GABA and recorded at Vhold -60, -40, -20, 0, 20, 40 mV

  23. GABAA IV Multi-hole Single-hole Erev Cl (avg) = -38 mV ± 4 mV, n=6 ErevCl (LJ corrected) = -51 mV ErevCl (theoretical) = -51 mV Erev Cl (avg) = -37 mV ± 3 mV, n=10 Erev Cl (LJ corrected) = -50 mV ErevCl (theoretical) = -51 mV

  24. GLuR5 Rise time multi-hole GLuR5 10 – 90% Rise time cursor (GLuR5 activated by 0.3 mMkainate) GLuR5 10 – 90% Rise time cursor (GLuR5 activated by 3 mMkainate) Rise time data for GLuR5 single hole; N.D

  25. GLuR5 Pharmacology GLuR5 currentsstimulatedwith 100 µM kainate and blockedwithincreasingconcentrations of CNQX GLuR5 currentsactivatedwby 100 µM kainate in increasingconcentrations of CNQX IC50 = 3.6 µM Rise time data are the same for

  26. GLuR5 IV Multi-hole GLuR5 currentactivated by 3 mMkainateand recorded at Vhold -60, -40, -20, 0, 20, 40 mV Erev (avg) = 16 mV ± 10 mV, n=14

  27. TE671 nAChR alpha1 Rise time α1 10 – 90% rise time cursor (α1 activated by 10 mMACh) Multi-holedata 10 – 90 % rise time for α1 currents Rise times areidentical for the twoQPatchtechnologies

  28. TE671 nAChR alpha1 Pharmacology α1 currentsactivated by increasingconc. of ACh (56 nM – 10 mM) Grouped Hill fit for α1 currentsactivatedwith ACh EC50 = 8.1 µM

  29. TE671 nAChR alpha1 IV X-mode Single-mode Erev(avg) = 8 mV ± 2 mV, n=5 Erev(LJ corrected) = -5 mV Erev(avg) = 8 mV ± 3 mV, n=11 Erev (LJ corrected) = -5 mV

  30. Screening on ligand-gated ion channels • New feature:Screening on ligand-gated ion channels is possible using the next software version from Sophion; will be released Fall 2010

  31. Summary Using X-mode technology Is the signal onsetslower? Is the biophysical/ pharmacologicalprofilecorrect? Is the success rate increased?

  32. Summary; Rise time data The current rise time from differentligandgated ion channels is comparable for multi-hole and single-hole data It is assumedthat the angle of the liquid front in the flow channel ”hits the cell” from aboveratherthan from the side

  33. Summary Using X-mode technology Is the signal onsetslower? Is the biophysical/ pharmacologicalprofilecorrect? Is the success rate increased?

  34. Summary Using X-mode technology Is the signal onsetslower? Is the thebiophysical/ pharmacologicalprofilecorrect? Is the success rate increased?

  35. Acknowledgement X-mode team Rasmus Bjørn Jacobsen Simon Pedersen Jesper Gerved Jens Henneke Patrick Juhl Jonatan Kutchinsky Morten Sunesen Søren Friis Ligand data Søren Friis Hervør Lykke Olsen Knirke Jensen Rasmus Bjørn Jacobsen Dorthe Nielsen Mette Christensen Jeffery Weber

  36. For more information WWW.SOPHION.COM

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