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 lawIon channel model

5. Current clamp Voltage clamp

6. Intracellular microelectrode technique R e << R in R in = 10 12 Ohm

8. 0.1 - 0.2  m Ag/AgCl 3 M KCl R e ~ 10 - 40 MOhm

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

12. drug 60 min20-60 min60 min 0 mV 20 mV 100 ms APA RP APD 50 APD 90 90% 50% V max Wash-outPre-incubation

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

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

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

16. Cell 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)

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

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

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

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

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

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

23. 1.Individual channels are either open or closed (no partial openings) 2.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 Properties of individual voltage- dependent sodium channels

24. 1.Individual channels are either open or closed (no partial openings) 2.Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. 3.Channel opening and closing is variable in duration and latency. Properties of individual voltage- dependent sodium channels The macroscopic sodium current

25. 1.The channels are either in open or closed state. 2.The channel openings are short events when compared with the macroscopic sodium current. 3.The time duration and latency of the channel openings are variable (case sensitive). Might happen to not open at all. 4.The open probability of the channels resembles with that of the macroscopic current. Properties of individual voltage- dependent sodium channels The macroscopic sodium current Summation of 300 recordings

26. 1.Individual channels are either open or closed (no partial openings) 2.Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. 3.Channel opening and closing is variable in duration and latency. 4.The overall probability of channel opening is similar to the total sodium current. Look at the sum of the currents from 300 trials. 5.Sometimes an individual channel doesn’t open even once. Summation of 300 recordings Properties of individual voltage- dependent sodium channels The macroscopic sodium current

27. 1.Individual channels are either open or closed (no partial openings) 2.Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. 3.Channel opening and closing is variable in duration and latency. 4.The overall probability of channel opening is similar to the total sodium current. Look at the sum of the currents from 300 trials. 5.Sometimes an individual channel doesn’t open even once. 6.Second openings are rare (because of inactivation) Summation of 300 recordings Properties of individual voltage- dependent sodium channels The macroscopic sodium current

28. Slowly inactivating K current channel (Ram & Dagan, 1987) 1.Individual channels are either open or closed (no partial openings). Sometimes more than one channel is in a patch. 2.Each channel opening is only a brief event compared to the total duration of the whole cell current. 3.Channel opening and closing is variable in duration and latency. 4.The overall probability of channel opening is similar to the whole cell current 5.Second openings can happen if there’s no inactivation. Similarly, individual potassium channels, calcium channels, and other channels can be studied by patch clamping

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

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

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

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

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

35. RsRs CmCm RcRc The whole-cell configuration

36. NaCl 144 NaH 2 PO 4 0.4 KCl 4 MgSO 4 0.53 CaCl 2 1.8 Glucose 5.5 HEPES 5 + I Ca blocker Intracellukar solution (mM) (for K currents) Extracellular solution (mM) (for K currents) K-aspartate 100 KCl 25 K 2 HPO 4 10, K 2 EGTA 5 K 2 ATP 3 MgCl 2 1 HEPES 10

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

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

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

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

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

43. Cell isolation - Ca 2+ - free perfusion - enzymatic digestion (collagenase) - mechanical separation

44. L- type calcium current (I Ca )

51. drug 5-10 min3-5 min10-15 min Wash-outPre-incubation Current-voltage (I-V) relationship L- type calcium current (I Ca )