Introduction to the Patch-Clamp technique

1. Danusa Menegaz, Ph.D
CCB/BQM/UFSC
Patch-Clamp:
Bioelectricity as a therapeutic target
Universidade Federal de Santa Catarina
Departamento de Bioquímica e Eletrofisiologia
NUBIOCEL (Núcleo de Bioeletricidade Celular)

2. Patch-Clamp
Voltage-clamp (mV) & Current-clamp (pA)
Allows the investigation of an unique channel
or multiple channels in individual cells;
Allows voltage-clamp to study currents (pA)
and current-clamp to study voltage (mV);
The principle of the method it is to isolate a
piece of the membrane electrically from the EM
and to register the ion flow through channels
with a high conductance electrodo;
Ion channels are involved in different
physiological functions like neurons signaling,
muscle contraction, cardiac rhythm, hormone
secretion and cell volume;

3. 1660 (Jam Swammerdam) electrical stimuli with with silver wire observed muscular
contraction in frogs;
1790 (Luigi Galvani) discovered ion channels and observed the nerve impulse
between motor neuron and muscle in frogs;
1850 (Hermann Helmholtz) velocity of propagation of a nerve impulse it was in
miliseconds;
1952 (Hodkin; Huxley), first register with intracelular electrodo – action potential in
giant axon of squid 0.5 mm; nobel prize in 1963;
1976 (Neher; Sakmann), patch-clamp discovered, single-channel, nobel prize in
1991;
History from eletrophysiology to Patch-Clamp

4. Patch-Clamp Set-Up
Isolated cellsTissue slices

5. Diseases involving ion channel function

6. Action potential in excitable cells

7. Pathological changes in the frequency of electrical
action potentials in neurons

8. Kv
Electrical activity of pancreatic β-cell

9. Electricity couple secretion in
Sertoli cells

10. Plasma membrane transport

11. Types of ion channels and activation factors

12. Electrochemical Gradient

13. Physiological solutions for the experiments
Extracellular media Intracellular media

14. Osmolarity of the IC and EC
OSMOMETER

15. Equipments for the patch-clamp technique
 Plataform:
 Anti-vibration tables;
 Mechanics and Optics:
 Microscope;
 Micromanipulators;
 Pipettes;
 Perfusion System;
 Eletronics:
 Faraday cage;
 Amplificators;
 Digitalizer and data acquisition;
 Noise;

16. Patch-Clamp Set-Up
Isolated cellsTissue slices

17. Electrical circuit

18. Microelectrodo – Pipette Puller

19. Steps to reach cell-attached configuration for the single-channel measurements

20. Patch-Clamp technique configurations
Single-channel
configurations
Whole-cell configurations

21. Current pulse (current-clamp) for the
voltage measurements (action potential)

22. Voltage pulses (Voltage-clamp) for current measurements
K+ and Cl- Channels
Na+ and Ca++ Channels

23. Sodium and Potassium channels – experimental solutions to avoid
other currents
K+ Channel Na+ Channel

24. Kv
Electrical activity of pancreatic β-cell

25. Kv channels in rat pancreatic islet

26. Chloride channels in Sertoli cells

27. Configuração Whole-cell (current-clamp)

28. Eletrophysiology and Molecular Biology

29. Thank you very much