LabAXON 5.

2 Workbook
Computer Simulation of
the Hodgkin & Huxley Equations
for the giant squid axon

Donald M. Bers, Ph.D.

Jose L. Puglisi, Ph.D.
Department of Pharmacology

University of California Davis

LabAXON Exercises on Excitable Membrane Properties

http://www.labheart.org
Objective: The purpose of these exercises is to help you better understand how axon cells generate
action potentials and how the action potential and the conductance of Sodium and Potassium are
related. This program (LabAXON) involves you interacting with a mathematical model of a Giant
Squid Axon that is based on experimental measurements carried out by Noble prizewinners
Hodgkin, Huxley, and Katz in 1952.
List of Abbreviations:
AP: Action potential; Vrm: Membrane voltage;
INa: Sodium current; GNa: Sodium conductance;
IK: Potassium current; GK : Potassium channel conductance;
IL: Leak current; GL : Leak conductance;
Note: the magnitudes of the ionic currents (e.g., INa, IK) are given as microamps/cm2, abbreviated
microA/ cm2 or A/ cm2. In several texts, the ionic currents are expressed as A/F. A farad (F) is
a measure of membrane capacitance and reflects cell size. This is a direct relationship between cell
size (cm2) and cell capacitance.

1. LabAXON: Introduction

Begin by opening the program and clicking on Start.

The page labeled Start will appear (name in top left corner)

The Help button will walk you through the menu. Click on the HELP and learn the
various functions especially how to measure with the cursors. Note that when it talks
about up/down and left/right arrows, it is referring to the red and blue diamonds on the
screen.
We will begin by exploring passive membrane properties before studying the action
potential thresholds.
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Action Potential Threshold:

Overview: The upstroke of the axon APs results from activation of large inward sodium
current (INa). This is also true for cardiac and skeletal muscle APs. INa activation will only
occur when membrane potential reaches the INa threshold voltage. In real cells, local circuit
currents act as the stimulus and depolarize membrane potential up to the INa threshold. In this
protocol you will examine the relationship between stimulus properties and AP initiation by
systematically increasing the amplitude of the stimulus until you reach the INa threshold and
initiate an action potential.
Procedure:
Go to Start MENU and click on RUN. You should see the same screen as below.

The ability to elicit an action potential is a function of both duration and amplitude of the pulse. A
pulse with short duration requires larger amplitude, and a pulse with small amplitude will require a
long duration. For example, a stimulus of 1 ms and 10A will not elicit an AP (see figure below).

Find the threshold for a 1 ms duration pulse by increasing the stimulus amplitude using the
amplitude knob.

Vary stimulus intensity according to Table 1. Repeat the measurments changing [K]o
to 6 mmol then set [K]o to the normal value and decrease [Na]o to 110 mmols.

Duration (ms)

Amplitude (mA) Amplitude (mA)

Control conditions [K]o =6 mmol

Amplitdue (mA)
[Na]o 110 mmol

5
4
3
2.5
2
1.5
1
0.8
0.6
0.3

Based on the above data construct the Intensity-Duration Curve

Intensity - Duration Curve

50

Amplitude (A)

40
30
20
10
0
0

Duration (ms)

3. Determination of refractory periods:

Overview: As you saw in #2 above, INa generates the AP upstroke. The channels first open
then they close (they inactivate). They remain inactivated throughout most of the action
potential until membrane potential returns to a negative value of approximately 70mV to
-80mV (The membrane repolarizes). As Vrm becomes increasingly negative an increasing
fraction of sodium channels recover from inactivation and the cell can generate another action
potential when stimulated. However, if the 2nd stimulus is applied before Vrm has recovered
to about 50mV, the cell will not fire an action potential (its refractory). APs of cardiac and
skeletal muscle also have a refractory period, produced by the same mechanism, except it is
much longer in duration.
Procedure: Go to the Stimulus Waveform Setup page by clicking on the wave icon.

Once on the waveform screen click on Two pulses and Exit

In this protocol you will assess refractory period by applying a second stimulus at various
times following the first action potential.
Click RUN and the Run Double Action Potential page will appear.

Now you are ready to apply two consecutive stimuli of the same duration and
amplitude to the cell.

Set the duration of both stimuli at 1 ms and the amplitudes at 15 A. Set the interval between
stimuli at 20 ms. You should see the stimuli in white, the AP in blue, the Na conductance in green,
and the K conductance in red. Repeat the procedure for intervals of 15 and 10 ms, and then decrease
the interval by 1 ms each time thereafter. At each interval, increase the stimuli duration keeping the
amplitude constant at 15 A. Then repeat the procedure keeping the duration constant at 1ms and
increase the amplitude. Fill the table below.

Interval (ms)

Duration (ms)
Amplitude = 15 A

Amplitude (A)
Duration = 1 ms

15
10
9
8
7
6
5
4
3
2

Based on the above data, build a graph with the interval on the x-axis and duration
and intensity both on the y-axis (left axis label Duration; right axis label Amplitude).

10

50

40

30

20

10

0
0

10

Interval (ms)

15

Amplitude (A)

Duration (ms)

Intensity - Duration Curve

4. Run Continuously protocol:

Overview: Although the cell responds in an all or none fashion, it is possible to elicit an AP
with a sub-threshold stimuli.
Procedure:
Set waveform to run continuously:

Set the DEFAULT Values, and EXIT. Then click RUN.

Decrease the stimuli to a sub-threshold value (i.e. 7.5 A, 1.5 ms). You will see that there is
no AP generated:

Without altering the characteristics of the stimuli (duration and amplitude), try to
generate an AP by increasing the frequency of stimulation.

Bonus: Set the duration and amplitude to 1 ms and 5 A, respectively, and try to
produce an AP.

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Voltage Clamp Experiments

There are three main protocols that are used to study the ionic channels under voltage clamp
conditions: IV-Set, Inactivation, and Recovery from inactivation. In this section you will
examine each of these protocols.
5. Current/Voltage relationship:
Overview: When the membrane is clamped to a particular voltage, a current flows through
the membrane. For each voltage, there is an associated current. By plotting the peak current
amplitude as a function of voltage the I-V set is generated. This plot provides the fingerprint
of the channel.

Procedure:
Go to WAVEFORMS, select Voltage Clamp by sliding the switch in the lower
left corner, the press the I-V set icon in the upper part of the screen, then EXIT.

From the Start window press Run, the I-V set screen will appear,

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Click on RUN to generate currents.

5a. When the membrane is depolarized, ALL currents that are available will
activate. The traces obtained are a combination of all of those channels. In order
to discriminate a particular current, we can do three things:
Block the other currents with a drug
Electrically inactivate the other currents, or
Replace one ion with another (e.g. Na with Choline, K with Cs, etc.).
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In our case, we will use TTX (tetrodotoxin) to pharmacologically block the Na

channels. Now the resulting trace is due solely to K channels.

By pressing I-V SET, the program will generate a current vs. voltage plot.

Close this window by pressing EXIT and you will be back on the I-V protocol screen.
13

5b Likewise, by blocking the Potassium channels the resulting traces will be due to Sodium.
On the I-V screen select TEA (Tetraethylammonium) a K channel blocker.
Run the program again. The traces will have the typical activation followed by the
inactivation of the channel.

By pressing I-V SET the program will plot the new current vs. voltage plot.
In this case the superimposition of K and Na channels.

Question: What would happen if you block both Na and K current?

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The second protocol that we are going to explore is the INACTIVATION of the channel
6 Channel Inactivation:
Overview: Once you depolarize a Na channel producing a current, a slower process takes
place: the INACTIVATION. This process closes the Na channel, making it inactive. To
remove the inactivation the membrane has to be repolarized to its resting value. Inactivated
channels cannot be activated to the conducting state until their inactivation has been removed.
The typical procedure to measure steady-state voltage inactivation consists of depolarizing the
cell by holding it at various voltages and then applying a test pulse. The difference in this
current amplitude is an index of what fractions of the channels were inactivated by the holding
potential

Procedure:
Go to Waveform, select Voltage Clamp by sliding the switch in the lower left
corner. Then press the Inactivation icon in the middle right part of the screen,
then EXIT

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From the Main Menu, go to RUN, the Inactivation screen will appear.
Within the Inactivation screen by pressing the RUN button all the traces will be generated

To understand better this phenomenon we will generate the traces one at a time
Press DEFAULT VALUES to insure the proper holding voltage (-100 mV)
Verify that the TEA button is ON because we are studying the Na channels
properties only
Set the iterations to 0. (At first we will do one pulse)
Press RUN

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The current that you obtained is the result of holding the membrane at 100 mv and depolarizing to
0 mV. If you move the cursor to the maximum amplitude the value will be indicated on the
corresponding field. Note: for a holding voltage of 100 mV the peak current is 3.09 mA/cm2.
Now repeat the experiment but with a holding potential of 80 mV, and measure the current.

The current peak value has decreased by the fact that some channels has been rendered inactive at
80 mV.

17

Repeat the measurements for holding potentials 100 to 0 mV in 10 mV steps.

Fill the following table
Holding Potential
(mV)
-100

Peak Current
(mA/cm2)

Normalized Value
(I/Imax)

-90
-80
-70
-60
-50
-40
-30
-20
-10
0
Based on the above values construct the Inactivation Curve

Inactivation Curve
1.00

0.75

6
0.50
4
0.25

2
0
-100

Normalized Current

-Peak Current (mA/cm2)

10

0.00
-80

-60

-40

-20

Holding Potential (mV)

This curve can also be obtained by setting the DEFAULT VALUES, running the program and
pressing the INACTIVATION PLOT.
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7 Recovery from Inactivation

Overview: As mentioned above each time the Na channel activates, it also undergoes a process of
inactivation. To remove that inactivation we have to repolarize the membrane back to its original
value. But this process takes time. We have to wait for the channel to recover. One way to
determine the rate of recovery is by applying two identical pulses with a varying interval and
measuring the different current amplitudes. (When the pulses are identical, the difference in current
amplitude is due to the recovery process).
Procedure:

Go to Waveform, select Voltage Clamp by sliding the switch in the lower left
corner. Then press the Recovery from Inactivation icon in the bottom part of the
screen, then EXIT

From the Main Menu, go to RUN, the Inactivation screen will appear.

19

Press DEFAULT VALUES and select TEA. (We are exploring the recovery from inactivation of
the Na channel). Click on RUN to generate the current traces.

As the interval between pulses is increased the second current amplitude recovers to its original
size. Clicking on the RECOVERY CURVE will generate the corresponding plot.

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Based on the above plot, what would be the maximum frequency to stimulate the Na
channel to obtain the same current amplitude?
Question: Why didnt we study the recovery from inactivation of the potassium channels?

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Single Channel Simulation

Overview: The purpose of this simulation is to realize that the macroscopic currents are the
result of the sum of individual events. Each single channel opens and closes in a stochastic fashion.
The current that is recorded during voltage clamp experiments is the cumulative of multiple single
channel events. The following exercises will illustrate the single channel activity for a Sodium (Na)
and a Potassium (K) channel, and their ensemble average.

Procedure:

Begin by clicking on the START Menu, then click on the Single Channel
(Na/K) Icon (Remember to close all previous simulations):

22

The following screen now appears:

This screen shows the state diagram for the K channel: 4 closed states and one open. That means the
K channel has to transition four times before reaching the open state. By pressing the K Channel
button you will switch to the other channel.

The state diagram for the Hodgkin and Huxley description of the Na Channel is a little bit more
complicated because assumes an independent inactivation mode. That means that simultaneously to
the activation process, the inactivation is also taking place (the parallel pathway on the diagram).
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Lets study first the single Na- channel behavior. In the previous screen press run and the following
screen will appear:

The upper graph shows the Voltage Protocol, The middle graph shows the Single channel activity,
and lower graph shows the Ensemble Average of the channels. The values on the graphs can be
measured by using the green cursors, and are displayed in the lower left yellow boxes.
Exercise 8 Measuring Sodium Current

Set the Holding potential to -90 mV, Step to voltage to 0 mV, Duration to 20
ms, and the Sweeps number to 50. Be sure that the channel is set to Na
(Orange button)
Click the RUN button, a screen similar to the one shown below will be
displayed

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Using the green cursors, measure the Voltage, Single current records and the absolute
maximum Ensemble Average. In our case, the values are 0 mV, -1.19 and -0.47 pA,
respectively.
(Note: as this is a stochastic process, it is unlikely that the exactly same values will be
obtained in your simulation)
Repeat the above procedure for various Step to voltages and record the values in the table
below. Holding voltage remains fixed at -90 mV.

Tip 1: At lower Step to voltages (-50, or -40 mV) perhaps you will not get a single opening on the
last sweep, and you will not be able to measure the current. If that is the case, set the sweep # to 1
and RUN the simulation until you get an opening and measure it.

Tip 2: You can change the simulation speed by moving the slide bar on the lower right panel

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Step to
Voltage
(mV)

Single Channel
Recording
(pA)

Ensemble
Average
(pA)

Single Channel
Recording
(pA)

Ensemble
Average
(pA)

[Na]o = 142 mM

[Na]o = 142 mM

[Na]o = 40 mM

[Na]o = 40 mM

-50
-40
-30
-20
-10
0
10
20
30
40
50
60
70
Based on the above Data construct the Current vs. Voltage Curve for both the Single Channel
Recordings and the Ensemble Average.

Sodium Channel
I-V Relationship

Current (pA)

-50

-30

-10

10

30

50

70

-1

-2

Voltage (mV)

Exercise 8b: Change the Na external concentration to 40 mM, repeat the measurements and
plots.

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Exercise 9: Measuring Potassium Current

Set the channel button to K CHANNEL (Green button)

Set the Holding potential to -90 mV, Step to voltage to 0 mV, Duration to 20
ms, and the Sweeps number to 50
Click the RUN button, a screen similar to the one shown below with be
displayed

Using the green cursors, measure the Voltage, Single current records and the maximum
Ensemble Average (towards the end of the pulse). In our case, the values are 0 mV, 1.90
pA, and 1.48 pA, respectively.
Notice that the K Channel opens more frequently as compared to the Na Channel.
Repeat the above procedure for various Step to voltages (from -60 mV to 60 mV) and record
the values in the following table:

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Step to
Voltage (mV)

Single Channel
Recording
(pA)

Ensemble
Average
(pA)

Single Channel
Recording
(pA)

Ensemble
Average
(pA)

[K]o = 4 mM

[K]o = 4 mM

[K]o = 20 mM

[K]o = 20 mM

-60
-50
-40
-30
-20
-10
0
10
20
30
40
50
60
Based on the above Data construct the Current vs. Voltage Curve for both the Single Channel
Recordings and the Ensemble Average:

Potassium Channel
I-V Relationship
3

Current (pA)

-60

-40

-20

20

40

60

-1

Voltage (mV)

Exercise 9b: Change [K]o to 20 mM, repeat the measurements and the plots.
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Exercise 10: Editing Equations

LabAXON 5.0 provides you with the capability of editing the gating equations ( and for
activation and inactivation). In these exercises you will explore the effects on changing the rate
constants for each process and see the effects at the single channel level and at the action
potential waveform as well.
Exercise 10a : Altering the K channel Activation
Run a control experiment under the single K channel screen.
Set the Holding potential at -90 mV, Step to 0 mV, Duration 20 ms and Sweep # 50.
You will obtain the following traces.

Click on the GATING FORMULAS button. The following screen will appear

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Change the Alpha n equation by typing -0.1, replacing the original -0.01 value. We just
increased the activation rate by 10 times. Press RUN, the new activation rate will be plotted.

Exit this window and return to the SINGLE CHANNEL simulation

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Run the same protocol than before, you will obtain a screen similar to the one below.

You can observe that the K current reaches its steady state value faster than before.
Lets see what are the consequences of this modification on the Action Potential waveform.
Click on the ACTION POTENTIAL button. The following screen will appear

There is no Action Potential!

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Click on the GATING EQUATIONS button and restore the DEFAULTS values
(Remember to press RUN!)

Now you will obtain the normal Action Potential!

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Question: Why a very fast K current makes impossible to generate an Action Potential?
The answer is that we need a net flow of charges through the membrane to depolarize. When the
Na channel opens, positive charges (Na+) flow inside of the cell, if at the same time the K
channel opens allowing positive charges (K+) to leave the cell, we got even and the membrane
does not depolarize. That why is so important a delay between activation of the Na channel and
activation of the K channel. Interesting concept, isnt it?
Lets see what happens when we increase the Beta n equation.

Go to the GATING EQUATION window and type 0.568 instead of 0.125 on the Beta n field.

Then press RUN to enter the modification.

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Go to the single channel simulation and run the same protocol than before.

You will notice that the ensemble current has dropped substantially, although the single channel
current remains the same. As the K channel leaves the open state more quickly the net result is a
smaller (or weaker) repolarizing current.
Lets see what happens with the Action Potential waveform

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This long AP is due to the fact that the K current is not able to provide enough depolarizing
current to terminate the AP.
Bonus exercises: Try to increase slightly Beta n to 0.56 and see the effects on AP.

35

Exercise 10b: Altering the Na channel inactivation.

The main characteristic of the Na channel is that undergoes to an inactivation process once it
has been activated. At the single channel level you can visualize that behavior as a burst of
opening at the beginning of the depolarizing pulse followed by a lack of activity at the end.
Go to single channel simulation and run the same protocol as before
Holding Voltage -90 mV, Step to 0 mV, Duration 20 ms, Sweeps # 50
Your screen will look like the one below.

Go to the GATING FORMULAS by clicking on the corresponding button

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Click on the SODIUM CHANNEL tab to obtain the Na gating equations.

Alter Beta h (inactivation) by typing 0.5 replacing the 1 in the numerator.
By doing so we are decreasing the rate by which the channel enters the Inactivation state.
(Remember to click on the RUN button to enter the modifications )
Return to the Single channel simulation and RUN the same protocol

First observe that the current trace lasts longer. It inactivates slower, and second the peak
ensemble current is bigger (why?).

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To make things worse now we are going to increase Alpha h. That implies that the channel
leaves faster the inactivation state.
Go to GATING FORMULAS and type 7 instead of 0.07.
By doing so we are almost canceling the inactivation process.

Run the single channel simulation and you will obtain traces similar to the ones below

The Na current now looks like a K current.

How would it be the shape of the AP?
Click on the ACTION POTENTIAL button to see the answer
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The Action Potential never repolarizes because the Na current does not inactive keeping the
influx of charge going through the cell membrane.
Now lets analyze the effect of altering the activation process.
Go to the GATING FORMULAS, restore DEFAULTS, and type -0.01 in the Alpha m field.
We are decreasing the rate by which the channel enters into the Open state.

Go to the single channel simulation and run the usual protocol

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By decreasing the activation rate the channel rarely opens, producing a very small net Na
current. What would it be the effect on the AP?

As you could have guessed no action potential is being generated.

The role of Na current is to initiate the action potential. When we decreased the activation
process the net Na current is diminished rendering it unable to start the AP.
A similar result can be achieved by altering (increasing) the Beta m gating parameter.

40

Measuring membrane capacitance (Cm)

Overview: Every cells membrane acts as a capacitor. The bigger cell the bigger capacitance.
Also, a bigger cell develops a larger current. In order to compare the currents elicited by
different cells it is necessary to normalize them by the size of the corresponding cell. In earlier
times, researchers reported the currents normalized by the area (mA/cm2). Today, the
capacitance value is used instead. In this section we will measure the capacitance of the
membrane by applying a rectangular pulse in the same way that Hodgkin and Huxley did in
their paper of 1952 .(Measurement of Current-Voltage Relations in the Membrane of the Giant
Axon of LOLIGO, Hodgkin, Huxley & Katz, J. Physiol 116, 424-448, 1952)
Procedure:
Go to the Start screen and click on the Capacitance icon (Cm) on the lower left side.

The Capacitance measurement screen will appear.

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Choose a particular axon, (e.g. Axon #8) and click RUN.

In the lower panel, the applied square pulse is shown. In the upper panel, the
corresponding current (yellow trace) is shown. The program also calculates the
integral of that current (red trace). We will use that value to calculate the
capacitance. Take note of that value (upper right side)
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Input the amount of charge in the lower left field

LabAXON will calculate the capacitance based on that value and the voltage
applied (30 mV in our example)
Press CALCULATE and the value of the membrane capacitance will be
estimated.
Repeat the same procedure with at least 5 different axons.

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Once you obtained a minimum of 6 values, press the CAPACITANCE PLOT button

Question: What can you infer about the relation between membrane area and capacitance?
44