study of ionic currents by the patch clamp technique andres soosaar andress

Study of Ionic Currents by the Patch Clamp Technique

Andres Soosaar

http://biomedicum.ut.ee/~andress

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

http://www.mmi.mcgill.ca/Dev/chalk/

Voltage clamp technique

• The voltage clamp method gives a possibility to hold the membrane potential on certain level

http://neuron.duke.edu/userman/ref/controlc.html

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

Patch clamp technique

http://www.nbtc.cornell.edu/Course/Lectures/Nineteen/ppframe.htm

http://www.nbtc.cornell.edu/Course/Lectures/Nineteen/ppframe.htm

Different possibilities for membrane patch

http://g1.ion.ac.cn/methods.htm

The simplified electric model of membrane

http://www.cnbc.cmu.edu/~bard/passive2/

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

http://imc.gsm.com/demos/hpdemo/program/section1/1ch6/1ch6line.htm

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The patch clamp circuit

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

http://medweb.bham.ac.uk/research/calcium/SupportFiles/Pclampfig.html

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

http://medweb.bham.ac.uk/research/calcium/SupportFiles/Pclampfig.html

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

http://medweb.bham.ac.uk/research/calcium/SupportFiles/Pclampfig.html

A typical neural action potential (AP)

http://cwx.prenhall.com/bookbind/pubbooks/silverthorn2/medialib/imagefold.html

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

http://www.thieme.de/elm/sim/patch2.html

Patch-Clamp Amplifier

• Power• Whole-Cell ParametersThat’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.

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

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

Oscilloscope

• There are automatic and “by hand” ways to present data

• Zoom

• A possibility to save data

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

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

Results

Ionic currents, pA Stim. (mV) Total

Na+ current (TEA+nifedipine)

K+ current (TTX+nifedipine)

Inward Outward Inward Outward Inward Outward 1 2 3 4 5 6

-3000

-2500

-2000

-1500

-1000

-500

0

500

1000

1500

2000

2500

3000

-70 -50 -30 -10 10 30 mV

pA

• TTX or tetrodotoxin blocks Na+ channels

• TEA or tetraethyl ammonium blocks K+ channels

• Nifedepin blocks Ca2+ channels