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CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES

CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES. NORBERT JOST, PhD. Electrical model of the membrane Standard intracellular microelectrode technique Voltage clamp technique Patch clamp technique. G=1/R. Ohm’s law. Ion channel model. Current clamp. Voltage clamp.

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CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES

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  1. CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES NORBERT JOST, PhD

  2. Electrical model of the membrane Standard intracellular microelectrode technique Voltage clamp technique Patch clamp technique

  3. G=1/R

  4. Ohm’s law Ion channel model

  5. Current clamp Voltage clamp

  6. Intracellular microelectrode technique Re<< Rin Rin = 1012 Ohm

  7. Ag/AgCl 3 M KCl Re ~ 10 - 40 MOhm 0.1 - 0.2 m

  8. The setup amplifier computer ingerlő A/D d e r Detected signal P 0mV Organ bath d: stimulating electrode 50 mV e: microelectrode r: referent electrode 100 ms P: preparation

  9. 20 mV 100 ms 0 mV 50% APA Vmax 90% RP APD50 APD90 Pre-incubation drug Wash-out 60 min 20-60 min 60 min

  10. Two microelectrode voltage clamp test potential voltage command holding potential The macroscopic sodium current

  11. The voltage-clamp circuit follow up amplifier voltage measure voltage command amplifier Current measure

  12. Patch-clamp: the special case of the voltage clamp

  13. Patch-clamp: the special case of the voltage clamp (1) Suck a small piece of membrane onto the tip of a glass micropipette (~ 1 µm in diameter) Cell

  14. Patch-clamp: the special case of the voltage clamp (2) “Gigaohm-seal” R > 1 GOhm Cell

  15. Patch-clamp: the special case of the voltage clamp (3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant. Cell

  16. Patch-clamp: the special case of the voltage clamp (3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant. closed open + + Cell

  17. Patch-clamp: the special case of the voltage clamp (3) Turn on the aimed potential the inside part of the pipette and keep it constantly by applying the voltage clamp technique. closed open open Cell

  18. Properties of individual voltage-dependent sodium channels voltage command 10 msec

  19. Properties of individual voltage-dependent sodium channels • Individual channels are either open or closed (no partial openings)

  20. Properties of individual voltage-dependent sodium channels • Individual channels are either open or closed (no partial openings) • Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. The macroscopic sodium current

  21. Properties of individual voltage-dependent sodium channels • Individual channels are either open or closed (no partial openings) • Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. • Channel opening and closing is variable in duration and latency. The macroscopic sodium current

  22. Properties of individual voltage-dependent sodium channels • The channels are either in open or closed state. • The channel openings are short events when compared with the macroscopic sodium current. • The time duration and latency of the channel openings are variable (case sensitive). Might happen to not open at all. • The open probability of the channels resembles with that of the macroscopic current. Summation of 300 recordings The macroscopic sodium current

  23. Properties of individual voltage-dependent sodium channels • Individual channels are either open or closed (no partial openings) • Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. • Channel opening and closing is variable in duration and latency. • The overall probability of channel opening is similar to the total sodium current. Look at the sum of the currents from 300 trials. • Sometimes an individual channel doesn’t open even once. Summation of 300 recordings The macroscopic sodium current

  24. Properties of individual voltage-dependent sodium channels • Individual channels are either open or closed (no partial openings) • Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. • Channel opening and closing is variable in duration and latency. • The overall probability of channel opening is similar to the total sodium current. Look at the sum of the currents from 300 trials. • Sometimes an individual channel doesn’t open even once. • Second openings are rare (because of inactivation) Summation of 300 recordings The macroscopic sodium current

  25. Similarly, individual potassium channels, calcium channels, and other channels can be studied by patch clamping • Individual channels are either open or closed (no partial openings). Sometimes more than one channel is in a patch. • Each channel opening is only a brief event compared to the total duration of the whole cell current. • Channel opening and closing is variable in duration and latency. • The overall probability of channel opening is similar to the whole cell current • Second openings can happen if there’s no inactivation. Slowly inactivating K current channel (Ram & Dagan, 1987)

  26. The configurations of the patch-clamp technique On-Cell Cell-Attached

  27. The configurations of the patch-clamp technique Inside-out patch On-Cell

  28. The configurations of the patch-clamp technique On-Cell Whole Cell

  29. The configurations of the patch-clamp technique Whole Cell

  30. The configurations of the patch-clamp technique outside-out patch Whole Cell

  31. The whole-cell configuration Rs Cm Rc

  32. Extracellular solution (mM) (for K currents) Intracellukar solution (mM) (for K currents) NaCl 144 NaH2PO4 0.4 KCl 4 MgSO4 0.53 CaCl2 1.8 Glucose 5.5 HEPES 5 + ICa blocker K-aspartate 100 KCl 25 K2HPO4 10, K2EGTA 5 K2ATP 3 MgCl2 1 HEPES 10

  33. The whole cell configuration Intracellular solution Patch-clamp amplifier Micropipette IBM PC + _ _ + _ + Extracellular solution _ _ + + + _ Cell _ + + _ + + _ _ 10 ms ... 5000 ms -20 mV ... +50 mV -40 mV

  34. The “run-down“ effect The ATP-sensitive potassium current

  35. The “run-down“ The L-type calcium current

  36. The configurations of the patch clamp technique Whole Cell, perforated patch Whole Cell - amphotericin-B - nystatin

  37. The “run-down” The L-type calcium current

  38. Cell isolation - Ca2+ - free perfusion - enzymatic digestion (collagenase) - mechanical separation

  39. L- type calcium current (ICa)

  40. L- type calcium current (ICa)

  41. L- type calcium current (ICa)

  42. L- type calcium current (ICa)

  43. L- type calcium current (ICa)

  44. L- type calcium current (ICa)

  45. L- type calcium current (ICa)

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