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Protocols of cardiac ion channels hERG, Ca ++ and Na +

CIPA project. Protocols of cardiac ion channels hERG, Ca ++ and Na +. Eunjung Park FDA/CDER/OND/DCRP. Summary of protocols. *. *. Access and seal resistance of manual patch clamp assays. *. Pipette (tip) resistance (access resistance, electrode resistance):

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Protocols of cardiac ion channels hERG, Ca ++ and Na +

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  1. CIPA project Protocols of cardiac ion channelshERG, Ca++ and Na+ Eunjung Park FDA/CDER/OND/DCRP herg

  2. Summary of protocols summary

  3. * * Access and seal resistance of manual patch clamp assays * • Pipette (tip) resistance (access resistance, electrode resistance): • < 5M in 67 out of 70 literatures (96%) including hERG, Ca++ and Na+ channels • Seal resistance: • 3 (* on graph) out of 70 protocols (4.3%) indicated its seal resistance as > 1G. • Series resistance compensation: • 35 out of 70 protocols (50%) indicated its series resistance compensation (minimum 60% and maximum 90%). summary

  4. Seal resistance in automatic patch clamp assays Rseal: Patch seal resistance, Rm: membrane resistance during whole cell recording, Ra: whole cell access resistance, Re: electrode resistance summary

  5. Extracellular buffer M: conventional manual patch clamp (mainly, Axopatch) A: Automatic patch clamp (): included in less than half of protocols summary

  6. Intracellular buffer M: conventional manual patch clamp (mainly, Axopatch) A: Automatic patch clamp, (): included in less than half of protocols summary

  7. Representative pulse sequences for a drug evaluation Depolarizing (0 to 70 mV, 1-4s) Repolarizing (-80 to 20 mV, 2-6s) hERG Vh=-80 mV Depolarizing (0 to 20mV, 100 to 500 ms) hCav1.2 Vh=-90 to -40 mV Depolarizing (-30 to 0 mV, 10-300 ms) Hyperpolarizing (-120 mV) hNav1.5 Vh=-150 to -80 mV Ref: JCE 2010, 21, 301 summary

  8. hERG assay herg

  9. Representative intracellular buffer solution UWM: University of Wisconsin at Madison herg

  10. Representative extracellular buffer solution UWM: University of Wisconsin at Madison herg

  11. Pulse sequences: potency * hERG enhancer herg

  12. Pulse sequences: Voltage dependent herg

  13. Pulse sequences: Channel activation * hERG enhancer herg

  14. Pulse sequences: Channel deactivation * hERG enhancer herg

  15. Pulse sequences: Channel inactivation herg

  16. Pulse sequences: Channel recovery from inactivation herg

  17. L-type Ca++ channel assay Ca channel

  18. Intracellular buffer composition of L-type Ca++ channel study Charge carrier /K blocker resistance internal Ca rundown chelating Cs-asp: Cs-aspartate, Cs-ME: Cs-methanesulphonate. TEA-Cl: tetraethylammonium chloride, CP-E (unit/ml): Creatine phosphokinase, Tris-P: tris-phosphocreatine, CP: creatine phosphate, MS: methanesulfonic acid. *CdCl2 (200 UM) added at the end of exp to block Ca current and leak calculation/**to test Ca standard, add just before recording with 0.2 mM cAMP and 3 mM CaCl2 Ca channel

  19. Extracellular buffer • Ingredients of extracellular buffer solution are quite similar between Ca channel and hERG assay except CsCl, TEA-Cl, and Choline Cl. • TEA (Tetraethylammonium): block K channel, formation and maintenance of giga seals • BaCl2= external charge carrier (to increase the amplitude of Ca channel), block other channels like Kir2.3 and Na during recording and maximize the L-Ca. Ca channel

  20. Voltage protocols for Ca++ channel ID49 ID93 inactivate the Na+ and T-type channels used to reveal state-dependent block augmentation in Cav1.2. Ca channel

  21. Pulse sequences: Potency Depolarizing (0 to 20mV, 100 to 500 ms) Vh=-90 to -40 mV Ca channel * Dr. January: no relevant articles

  22. Pulse sequences: Voltage dependent • Kinetic study of L-type Ca channel - limited to voltage dependent/ state-dependent/use-dependent protocols • Kinetic study in HT assay- limited. Ca channel

  23. Na+ channel Na channel

  24. Intracellular buffer of Na+ channel assay Charge carrier /K+ blocker Charge carrier /Ca++ blocker Cs-asp: Cs-aspartate, TEA-Cl: tetraethylammonium chloride, CP: creatine phosphate Fluoride: introduction of fluoride anions into the cell produced an irreversible block of calcium current. Na channel

  25. Extracellular buffer • NMDG (N-methyl-D-glucamine), lower mobility, produce series resistances larger (by about 30-50%) than Cs. • L-AA: L-aspartic acid Na channel

  26. Pulse sequences: Potency Depolarizing (-30 to 0 mV, 10-200 ms) Hyperpolarizing (-120 mV) holding (-150 to -80 mV) * Two step pulse for tonic and phasic block Na channel

  27. Pulse sequences: Voltage dependent Na channel

  28. Pulse sequences: channel kinetic protocols Na channel

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