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Study of Ionic Currents by thePatch Clamp Technique

Andres Soosaar

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

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

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Methods to study cellular

bioelectricity Main design: Cell --

Electrodes AmplifierVoltmeter orGalvanometer Printer

There several to ways tolocate electrodes to cell:

Using of extracellular orintracellular electrodes.

The extracellularelectrodes are usually

Ag/AgCl wires The intracellular

electrodes are small tip(~1 m) glass pipettes

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

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Voltage clamp technique

The voltage clamp

method gives a

possibility to hold

the membranepotential on certain

level

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

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The patch clamp method

Certain membrane region is electrically separated fromneighbouring regions by gigaseal (R >109)

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 andmembrane < 1nm

The patch clamp method gives a possibility to measurecurrents going through a single or few ion channels

As ion channels are in the membrane of every cell, thepatch clamp technique is usable to study of any cell

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Patch clamp technique

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

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

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Different possibilities for membrane

patch

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

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The simplified electric model of

membrane

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

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

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The time constant

Time constant is the rise

time of potential to 62.7

% of maximal value. For neurons is ranged

from 5 to 50 ms

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

mm

t

me

CR

eRIV

)1(

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

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The patch clamp circuit

The difference between Vm and Vcmd is corrected

by injecting Vo down the micropipette. Thisdepolarises the membrane and voltage gatedchannels open. The current flowing through asingle channel is Ip

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

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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 ofcurrent measurement, and the background noise level.Usually Rf is 5-10 G

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

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A typical neural action potential

(AP)

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

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

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The Setup of SimPatch

Patch-clamp amplifier

Stimulator or pulsegenerator

Oscilloscope

Cell(s), electrodes andheadstage amplifier aremissing on the screen

The bottom line buttonsare for management of

and to use additionalfacilities of the virtualsystem

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

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Patch-Clamp Amplifier

Power

Whole-Cell Parameters

Thats the system to reduce membrane capacitive

currents. Adjusting Capacitance and SeriesResistance knobs you can find the situationwhen capacitive currents are reduced (NB!

Apply single puls from generator and after that

adjust knobs).As capacity C=SCm , Cm=1F/cm

2 , there ispossbile to calculate cell surface area.

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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

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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

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Oscilloscope

There are automatic and by hand ways

to present data

Zoom

A possibility to save data

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Additional modules

Settings: Dont change default settings

Solutions: It gives an overview aboutdifferent mediums usable in different

experiments Cell selection

Edit stimulus properties

Data analysis: use special cursors tomeasure ionic currents and dont save anydata

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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 thecapacitive 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 theminto table)

Apply different solutions to separate ionic currents through differentchannels.

Conclusions: It should contain summary about ionic currents andchannels of the selected cell.

Choose another cell for study

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Results

Ionic currents, pAStim.(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 30mV

pA

TTX or tetrodotoxin

blocks Na+ channels

TEA or tetraethyl

ammonium blocks K+channels

Nifedepin blocks Ca2+

channels