Electrophysiological Techniques I

Question 1

Osmotic pressure favours osmosis of water into cells due to the high intracellular [protein]. How is cell volume controlled so that cell lysis does not occur? What is the consequence of this for electrochemical gradients in the cell?

Answer 1

The Na⁺/K⁺ ATPase produces an uneven distribution of Na⁺ and K⁺ to reduce osmotic pressure and prevent cell lysis.

This leads to creation of electrochemical gradients for Na⁺ and K⁺.

Question 2

Describe the movement of K⁺ ions in and out of the cell at rest.

Answer 2

At rest, K⁺ flows out of the cell until -ve charge prevents it.

Question 3

What does the Nernst equation describe?

Answer 3

The membrane potential created by K⁺ when the membrane is freely permeable to K⁺, = -90mV

Question 4

What is Ohm’s law?

Answer 4

Voltage (V) = Current (I) x Resistance (R)

Resistance = 1/Conductance; R = 1/G

Current = Conductance x Voltage; I = G V

Question 5

How does Ohm’s law relate to cell electrophysiology?

Answer 5

Electrophysiology involves the recording of I, G and V to measure the activity of ion channels

Question 6

Name the 3 main microelectrode techniques.

Answer 6

1. Extracellular recording
2. Intracellular recording
3. Voltage-clamp recording

Question 7

Explain how extracellular recording of electrical activity is conducted.

Answer 7

• 2 microelectrodes placed outside cells; a ground (reference) electrode and recording electrode.
• Records cell-induced membrane potential changes between 2 electrodes - e.g. action potentials.
• Single cells - single-unit recording.
• Group of cells - multi-unit recording.

Question 8

Explain how intracellular recording of electrical activity is conducted and describe 2 uses.

Answer 8

• 1 microelectrode placed inside, 1 ground electrode placed outside.
• Records membrane potential across membrane of single cell.
• Allows manipulation of external solutions but does not alter intracellular composition.

Uses:

1. Record electrical responses to external stimuli - e.g. neurotransmitters.
2. Measure changes in conductance - opening/closing of ion channels.

Question 9

Explain the basic principles of voltage-clamp recording using an example of a neuron “clamped” at -20mV.

Answer 9

• 1 microelectrode measures voltage and is “clamped”.
• 1 microelectrode applies current to and from the cell.

1. E.g. step a neuron from -70mV to -20mV
2. Amplifier provides current to “clamp” at -20mV
3. Current applied = currrent through Na⁺/K⁺ channels at -20mV

Question 10

Hodgkin, Huxley and Katz developed the voltage-clamp technique and used it to discover what?

Answer 10

They discovered that the initiation of an action potential involves Na⁺ ions.

Question 11

Explain the advantages of using intracellular recording over extracellular recording of electrical activity.

Answer 11

• Extracellular recording detects electrical activity but doesn’t tell you anything about it - essentially it just detects action potentials.
• Intracellular recording allows you to measure the conductance across a membrane and measure the electrical response to external stimuli.

Question 12

Why is voltage-clamp recording such a powerful technique in electrophysiology? What useful information does it give us?

Answer 12

Voltage-clamp recording allows us to measure membrane current produced by opening of ion channels at a specific membrane potential.