1. Study of Ionic Currents by the Patch Clamp Technique
Andres Soosaar

2. Methods to study cellular bioelectricity
Main design: Cell -- Electrodes – Amplifier – Voltmeter or Galvanometer – Printer
There several to ways to locate electrodes to cell:
Using of extracellular or intracellular electrodes.
The extracellular electrodes are usually Ag/AgCl wires
The intracellular electrodes are small tip (~1 μm) glass pipettes

3. Voltage clamp technique
The voltage clamp method gives a possibility to hold the membrane potential on certain level

4. The patch clamp method
Certain membrane region is electrically separated from neighbouring regions by gigaseal (R >109 Ω)
There are several ways to get a membrane patch
Often voltage clamp and patch clamp are combined into one method
Glass pipettes serve as electrodes for patch clamp and by the gigaseal the distance between pepette tip and membrane < 1nm
The patch clamp method gives a possibility to measure currents going through a single or few ion channels
As ion channels are in the membrane of every cell, the patch clamp technique is usable to study of any cell

5. Patch clamp technique

7. Different possibilities for membrane patch

8. The simplified electric model of membrane

9. The time constant τ
Time constant is the rise time of potential to 62.7 % of maximal value.
For neurons τ is ranged from 5 to 50 ms

10. The patch clamp circuit
The amplifier compares the membrane potential (Vm) to the new command potential (Vcmd) specified by the operator as -20mV

11. The patch clamp circuit
The difference between Vm and Vcmd is corrected by injecting Vo down the micropipette. This depolarises the membrane and voltage gated channels open. The current flowing through a single channel is Ip

12. The patch clamp circuit
The current passing through all the channels (Ip) flows through the circuit and is measured as a voltage change.
Vo= -Rf· Ip + Vcmd -Ip · Rf = Vo- Vcmd
Rf (feedback resistor) determines the sensititvity, range of current measurement, and the background noise level. Usually Rf is 5-10 GΩ

13. A typical neural action potential (AP)

14. The Setup of SimPatch Patch-clamp amplifier
Stimulator or pulse generator
Oscilloscope
Cell(s), electrodes and headstage amplifier are missing on the screen
The bottom line buttons are for management of and to use additional facilities of the virtual system

15. Patch-Clamp Amplifier
Power
Whole-Cell Parameters
That’s the system to reduce membrane capacitive currents. Adjusting Capacitance and Series Resistance knobs you can find the situation when capacitive currents are reduced (NB! Apply single puls from generator and after that adjust knobs).
As capacity C=S·Cm , Cm=1μF/cm2 , there is possbile to calculate cell surface area.

16. Patch-Clamp Amplifier
Display for different currents and voltages, Vm shows membrane potential
The Mode switch should be in V-clamp position
Gain shows the level of amplification
Connections

17. Pulse generator
There are 2 ways to deliver impulses: single and family (6 impulses) of impulses
Output 1 connects stimulator with specimen
Output2 connects stimulator with oscilloscope

18. Oscilloscope There are automatic and “by hand” ways to present data
Zoom
A possibility to save data

19. Additional modules Settings: Don’t change default settings
Solutions: It gives an overview about different mediums usable in different experiments
Cell selection
Edit stimulus properties
Data analysis: use special cursors to measure ionic currents and don’t save any data

20. How to perform experiment?
Switch on all 3 devices
Choose a cell for experiment
Check solutions box and choose at first standard solutions
Apply the single impulse and adjust C and R to reduce the capacitive current of the membrane. If you are interested, you can calculate cell surface area
Apply the impulse family to cell
Analyse data curve by curve (different Vm values and record them into table)
Apply different solutions to separate ionic currents through different channels.
Conclusions: It should contain summary about ionic currents and channels of the selected cell.
Choose another cell for study

21. Results TTX or tetrodotoxin blocks Na+ channels
TEA or tetraethyl ammonium blocks K+ channels
Nifedepin blocks Ca2+ channels