SCHOOL OF

BIOMEDICAL
SCIENCES

BMS1031 – Medical
Biophysics

“MEMPOT” –
Computer Simulation
of Membrane Potential
Measurements

STUDENT
WORKSHEET 2024
A/Prof. Yan Tat Wong
Dr. Ari Pinar
Dr. Michael Leung
Prof. Kristian Helmerson
LEARNING OBJECTIVES
1. To explore the effect varying the extracellular potassium ion concentration ( [K+]o ) has on the resting membrane
potential and to plot the relationship between resting membrane potential and [K+]o
2. To explore the effect varying the external sodium concentration ( [Na+]o ) has on the peak of the action potential
and to plot the relationship between action membrane potential and [Na+]o
3. To analyse clinical case studies relating to alterations in resting membrane potential.

INTRODUCTION
“MEMPOT”© is an interactive graphical simulation program that is designed to show how cell membrane potentials
are measured with microelectrodes in the laboratory situation. As traditional lab-based recordings of membrane
potential can be prohibitively expensive, the “MEMPOT”© simulation shows how the membrane potential depends
on the external potassium, [K+]o and external sodium, [Na+]o concentrations and the relative permeability of the
membrane to these ions. The program simulates membrane potential measurements in any excitable cell permeable
to K+ and Na+ ions (for example, neurons or muscle cells).

To record a cells membrane potential, a technique called “patch clamping” is used. A hollow glass micro-pipette is
filled with an electrolyte solution and a recording electrode. This is connected to an amplifier that can record the
voltage on the electrode. A second reference or ground electrode is placed in the dish or container that the cells are
in. The pipette is slowly advanced until it touches the surface of the cell membrane (step 1). This is very tricky and
requires very stable micromanipulators. If you advance too quickly or too far, then the cell will be killed. If you don’t
advance far enough then you won’t make contact with the cell and your recordings will be incorrect. Once the pipette
makes contact with the surface of the cell membrane, you can now record from the membrane channels underneath
the pipette (step 2). This is called a cell-attached patch recording. Alternatively, suction can be applied to the pipette
and the cell membrane is sucked up into the pipette and eventually ruptures the membrane without killing the cell.
Now you can record from the whole cell (step 3, whole-cell patch recording).

Fig 1. The steps involved in patch clamping. 1) Advance the micropipette, 2) get very close to the cell membrane,
and 3) suction until the cell membrane ruptures and you can now record inside the cell.
 Fig 2. An image of a cell that has been patched with the glass micropipette visible on the right of the image.

In this practical you will simulate a number of microelectrode experiments across two exercises. In the first of the
exercises we will explore the effect varying the extracellular potassium ion concentration ( [K + ]o ) has on the resting
membrane potential (RMP). You will then apply the concepts of the Nernst and Goldman–Hodgkin–Katz (GHK)
equation by plotting resting membrane potential against [K+ ]o.

In the second optional exercise (run online) we will again build on concepts introduced in the lecture and workshop
by exploring the effect varying the external sodium concentration ( [Na+ ]o. ) has on the peak of the action potential.
In each of these sessions we will explore a range of physiological case scenarios where we will examine the
influence varying certain physiological parameters (i.e., ionic concentration) have on influencing the resting
membrane potential.

The Mempot 1.2 shortcut is located on Monash MOVE or via the direct link on Moodle.

Main Title and Documentation Screens

The program starts up with a Welcome screen. After viewing this, click on the ‘Close’ button (or press
Enter) to close the Welcome screen and go to the Main Menu screen. Choosing Introduction will
then result in additional screens of instructions and information.
PROGRAM OPERATION:
The sequence of operations is normally:
(1) Introduction – Hypothetical Bilayer
(2) Resting Potential Experiment – Show Report – Print – Graph Resting Potential Results –
Show Report – Print
(3) Action Potential Experiment – Show Report – Print – Graph Action Potential Results –
Show Report – Print

For the purposes of this practical we will focus on (2) Resting Potential Experiment.
 START 1st
HERE
3rd

2nd

Last

Fig. 3. The main menu options available for the Membrane Potential Simulation program, MemPot 1.2.
 EXERCISE 1
RESTING MEMBRANE POTENTIAL
MEASUREMENTS
1. From the menu, open the “Resting Potential Experiment”. This exercise illustrates the effect of varying the
external (extracellular) potassium concentration, [K+]o on the resting membrane potential.

2. Choose “Start Experiment”. Begin by entering a [K+]o = 0.5 mmol/L. To do this, enter the value (e.g. 0.5) into the
box and then click “ok”. You should obtain measurements of the resting membrane potential in at least 6
different potassium concentrations from 0.5 to 150 mmol/L. (so, 0.5, 1.0, 5.0, 10.0, 20.0, 40.0, 100.0 and 150.0).

Fig 4. Experimental setup showing the main electrode screen of the MemPot program just after a solution concentration
has been chosen. Where necessary, the cells will be cleared and the electrodes repositioned and arrow-keys
highlighted. Any previous averaged values of measurements in other solutions would be listed in the bottom right panel,
as shown. The internal electrode can then straightaway be moved by mouse control with the 4 Direction buttons (or
Arrow keys of your keyboard) and stopped by the Stop button (or the End key). The electrode can be withdrawn from
a cell with the [Up] button.
 3. Use the arrow keys on your keyboard to sample each of the 5 cells. Put the electrode into the cell, but be careful
not to rupture the cell. For a successful impalement, only the tip of the microelectrode should enter the top
section of the cell (within half a radius of the centre). A sideways or glancing impalement will result in a 0 value
of membrane potential being recorded (and not included in any averaged results). After an impalement, the
electrode is withdrawn by clicking the [Up] button before another cell is impaled. Once a cell has been impaled,
its colour will be changed and it will become cross-hatched (not unlike muscle cells that tend to go opaque when
damaged). This has the added advantage that it clearly indicates which cells have been recorded from. from.
The value of the membrane potential is then printed on the centre panel and a simulated oscilloscope trace
(with simulated random electrical noise) is displayed in the left panel (Fig. 4).

4. The membrane potential will appear on the left of the screen (V m). Sample each of the 5 cells to get an average
resting potential (Vav) and an estimate of error (SEM) at each [K]o. Enter this data into Table 1 below, along with
the internal and external cation concentrations provided to you.

5. Once a reasonable range of [K+]o has been investigated, click “Finish Experiment” and return to the “Menu”.

6. Open “Graph Resting Membrane Potential Results”. This part of the practical requires you to re-enter the data
you have just recorded into the program (“Enter/Edit Data”), which will then generate a graph of resting
membrane potential against [K+]o. .

7. Plot a curve of the relationship between resting membrane potential and [K +]o. The program will plot the
predicted curve based on the Goldman-Hodgkin-Katz (GHK) equation for each permeability ratio you have
entered. The objective is to obtain the curve that best fits the data. You should then obtain an estimate of
the relative Na+ to K+ permeability by trial-and-error entry of values for PNa/PK. This tells you how permeable
the cell is to Na+ to K+ when the cell is at rest.

8. Once you have found the curve based on the GHK equation (red), you should also have the program plot the
ideal curve (based on the Nernst equation, blue dotted) that would be predicted if Na+ were impermeant.

• The Nernst Potential for an ion (be it Na+, K+, Cl-, etc.) predicts the resting membrane potential (RMP; VM) that
would result if membrane was permeable to only one type of ion. More accurately, the Nernst potential value
(equilibrium potential; Em) for a SINGLE ion corresponds to the membrane potential at which the electrical
gradient exactly opposes the concentration gradient. For K+, this would look like,

RT [K + ]o E : membrane potential
m

Em = ln + R: the gas constant (8.315 J mol K )

–1 –1

zF [K ]i T: absolute temperature (20 °C = 293 K)

+
z: charge on the ion (z=1 for K )
–1
F: Faraday’s constant (96485 C mol )
+ + +
[K ] , [K ] : K ion concentrations outside and inside cell
o o
ln: natural logarithm

Substituting appropriate values for R,T, z & F for a movement of a cation at 37 °C the Nernst Potential for an ion; E
m
(millivolts) is calculated from,

+
[K ]o
Em = 61 log 10
[K + ]i
Table 1: INSERT TABLE CAPTION

RMP SEM
[K+]o [K+]i [Na+]o [Na+]i (mV)
Nernst values
(mV)

Resting Potential Data

[K] O (mM; Log scale)
0.1 1 10 100 1000
20

-20

-40

(mV)
-60

-80

-100

-120

-140

Figure 1: INSERT FIGURE CAPTION

Worksheet Questions for Exercise 1
1. Comparing the Nernst equation to the GHK equation, how many ions does the GHK equation take into account?

2. Plot your obtained values from Table 1 into Figure 1. This should look like the figure you generated in the “Graph
Membrane Potential Experiment”. Draw on to the graph the curve of best fit.

3. Calculate the Nernst Potential for K+ ions and write these in Table 1. Show your working for at least one K +
concentration in the space below.

4. What effect does changing the concentration of [K]o have on the membrane potential of the cell? Does
increasing [K]o depolarise (make Vm more positive) or does it hyperpolarise the cell (making Vm more
negative)?

5. Plot these values on Figure 1 and draw on to the graph the ideal Nernst equation curve.

6. You now have two curves on your graph. One is the ideal curve for resting potential based on Nernst’s equation.
The other is the based on the data you generated in the Mempot simulation, which is fitted with the GHK
equation taking into account both Na + and K+. Describe how these curves differ visually at any given [K] o.
7. Explain the reasons why these differences exist.
EXERCISE 1 CLINCAL SCENARIO – TRANSLATION TO BIOLOGICAL SYSTEMS

Hyperkalemia

Hyperkalemia is a physiological condition characterised by elevated level of potassium (K+) ions within blood serum.
In a healthy adult normal blood serum K+ levels are between 3.5 and 5.0 mmol/L with levels above 5.5 mmol/L
defined as hyperkalemia. Even at these elevated levels typically this results in no symptoms, however in more
severe circumstances may result in muscle weakness, muscle pain and an abnormal resting heart rate (referred to
as a cardiac arrhythmia).

1. Using resources available to you (textbooks, notes, Internet) research and develop an understanding of the
physiological basis of hyperkalemia. Specifically, provide an understanding of how abnormally elevated blood
serum K+ levels can lead to abnormalities in the resting membrane potential
2. The image below shows a typical action potential. On the image below draw a curve of how you would expect
to observe an action potential waveform/trace from an individual with hyperkalemia.

3. Describe how hyperkalemia impacts a neurons ability to produce typical action potentials – we will explore action
potentials in greater detail in Exercise 2.

4. EXTENSION QUESTION: From your acquired understanding of how hyperkalemia leads to abnormalities in
the resting membrane potential, describe how hyperkalemia may result in cardiac arrhythmias.
EXERCISE 2 (optional extension activity)
ACTION POTENTIAL MEASUREMENTS

We will now investigate the effect of altering the external sodium concentration, [Na+]o. on the peak of the action potential.
The overall procedure is very similar to that already described for resting potential measurements (Exercise 1), however,
in this exercise, you are asked to adjust the external sodium concentration ( [Na+]o ) instead. Now, the [K+]o concentration
is held constant (e.g., at 5.5 mmol/L) and the concentration of impermeant choline [Cl-]o is automatically adjusted to
maintain a constant ionic strength It may again be assumed that the internal [K+]i and [Na+]i concentrations remain
constant (e.g., at 150 and 15 mmol/L respectively) and that the anions are impermeant. When the internal electrode
successfully impales a cell, the resting potential is again printed out and a simulated oscilloscope trace displayed. The
action potential is then activated by clicking on the OK button when prompted in the pop-up menu.

1. Open the “Action Potential Experiment”. This exercise allows you to investigate the effect of varying the external
sodium concentration, [Na+]o. on the peak of the action potential.

2. Choose “Start Experiment”. Begin by entering a [Na +]o = 5.0 mmol/L. To do this, enter the value (e.g. 0.5) into
the box and then click “ok”. You should obtain action potential measurements in at least 6 different sodium
concentrations from 5.0 to 150 mmol/L. (so, 5.0, 10.0, 20.0, 40.0, 100.0 and 150.0).

3. Again, sample each of the 5 cells to get an average action potential (V av) and an estimate of error (SEM) at
each [Na+]o. Enter this data into Table 2, along with the internal and external cation concentrations. Also enter
the values for choline (chol) – this is a positively charged molecule that does not cross the cell membrane.

4. Once a reasonable range of [Na+]o has been investigated Click “Finish Experiment” and return to the “Menu”.

5. Open “Graph Action Potential Results”. Again, you need to re-enter the data to generate a graph of action
membrane potential against [Na+]o.

6. Plot a curve of the relationship between action membrane potential and [Na+]o. The program will plot a predicted
curve (based on the Goldman-Hodgkin-Katz equation) for each permeability ratio entered. The objective is to
obtain the curve that best fits the data. You should then obtain an estimate of the relative Na+ to K+ permeability
by trial-and-error entry of values for PNa/PK.

7. Once you have found the curve of best fit (based on the GHK equation), you should also have the program plot
the ideal curve (based on the Nernst equation) that would be predicted if K + were impermeant.
Table 2. INSERT TABLE CAPTION

[K+]o [K+]i [Na+]o [Na+]i [Chol]o Peak of SEM Nernst

AP (mV) Value
(mV)

Action Potential Data

[Na] O (mM; Log scale)
0.1 1 10 100 1000
80

60

40

20
(mV)
0
m

-20

-40

-60

-80

-100

Figure 2. INSERT FIGURE CAPTION

Worksheet Questions for Exercise 2

1. Plot your values from Table 2 into Figure 2. This should look like the Figure you generated in the “Graph Action
Potential Experiment”. Draw on to the graph the curve of best fit.

2. Calculate the Nernst Potential for Na+ ions and write these in Table 1. Show your working for at least one
concentration in the space below.

3. Plot the Nernst values on the graph and draw the ideal Nernst equation curve for Na +.

4. What effect does changing the concentration of [Na+]o have on the overshoot of the action potential? Does
increasing [Na+]o increase or decrease the overshoot?

5. Again, you now have two curves on your graph which show a change in action potential amplitude dependent
on the [Na+]o. One is the ideal curve for action potential based on Nernst’s equation for Na+. The other is the
based on the data you generated in the Mempot simulation, which is fitted with the GHK equation. Describe
how these curves differ visually at any given [Na+]o.

6. Explain the reasons why these differences exist.

7. How does the relative permeability of the membrane to Na + and K+ throughout the course of the action potential
change compared with that at the resting membrane potential? Use your lecture notes to answer this question.

EXERCISE 2 CLINCAL SCENARIO – TRANSLATION TO BIOLOGICAL SYSTEMS

Liddle’s syndrome

Liddle’s syndrome is a rare form of hypertension associated with hyperactive epithelial sodium channels (ENaC)
expressed in certain epithelial tissues including the lungs, kidneys and the colon
1. Using resources available to you (textbooks, notes, Internet) research and develop an understanding of the
physiological basis of Liddle’s syndrome. List the most common symptoms of Liddle’s syndrome below.

2. Describe how the physiological process of hyperactive ENaC’s relates to the symptoms of Liddle’s syndrome
(listed above).
3. Amiloride is the first-line treatment for Liddle’s syndrome. Describe Amiloride’s site/s of action, AND discuss
how administration of Amiloride influences the resting membrane potential of epithelial cells
Assessment activity

BMS1031: Bioelectricity post lab assessment

Task: The purpose of this assessment is to help you contextualise how the understanding of biophysical
principles can allow you to better operate in other fields. You are to pick a disease or disorder that affects
bioelectricity in the body and create a video where you explain the biophysical underpinnings of the disease
or disorder. We would like you to put yourself in the role of a healthcare professional or expert in medical
biophysics and explain the following key points in lay terms to a patient:
1. The symptoms that the patient is experiencing,
2. The underlying biophysics that are causing the symptoms, with reference to how action potentials
may be affected, the cell membrane voltages and any other changes of interest, and
3. Possible treatments or cures and how these affect the patient’s bioelectricity.
You should refer to your notes for guidance and information and may use one slide to help with the
explanation i.e., annotate or adapt diagrams from your lab notes. Some examples include leg cramp after
exercise, Liddle’s syndrome, tetrodotoxin from eating puffer fish, or Multiple Sclerosis.

Submission:
• Record a video answer of 1 minute ± 30 seconds.
• Submit the mp4 file in Moodle
• The speed of videos cannot be artificially changed
• Recorded voice must be clear
• Your face should be visible on the video
• A maximum of 1 slide can be shown in the video
Marking Criteria (see below)
• Analysis, Synthesis & Understanding: The video content is entirely relevant to the topic
• Structure, Organisation & Communication: Information is presented in a logical sequence, and is
presented in a way that is understandable by non-expert
• Creativity: The video is highly engaging, and creativity is demonstrated in the video concept and
production
• Formatting: The video fulfils all of the formatting criteria, e.g., length, file type
Video recording
You can use any method to record your video answer. We recommend using the Desktop Zoom platform by
which you can share your whole screen while the camera and audio are on. To do that, you need to first sign
in to Zoom using your ID/pass, then press New Meeting tile under the Home tab. If you are using a slide you
should share your whole screen by pressing the green button named Screen Share, beside which the Record
button is located, both at the bottom of the Zoom window. When you are ready to record your answer, press
the Record button and select “Record on this Computer”. When finished, press Stop recording and then exit
from Zoom by pressing the “End” button located at the bottom right-hand corner. Zoom will convert your
recording to an mp4 which will be saved in the Zoom folder located in Documents. This .mp4 recording file is
the only thing you need to submit on Moodle. If you do not have a microphone/video camera on your
desktop/laptop, you can use a smartphone to join the zoom session started on the desktop where the
recording is set up. Useful information can be found here:
https://www.monash.edu/learnhq/build-digital-capabilities/use-video-streaming-tools/video-
assessments/record

How to submit your video?

The mp4 file should be directly uploaded to the submission box on Moodle. If, in any case, you are unable
to upload the file to Moodle, you may submit them to Panopto and link it to Moodle. Instructions on how to
do this can be found here: https://www.monash.edu/learnhq/build-digital-capabilities/use-video-streaming-
tools/video-assessments/submit

FAQ
1. Would I be marked on the quality of the video?
The resolution of the video will not be assessed if all visual aids just as graphs are legible.
2. Do I need to submit a script/written answers?
No script of answers to this week's lab needs to be submitted but the notes you take will help in the
assessment as well as the final exam.
3. I don’t know how to edit a video. What should I do?
Instructions on how to film a video are above. We will not be assessing video editing and as the video is
short (under 1.5 minutes) you should be able to film this in one shot.
4. Do I need to provide references for the information/image that I use for the video?
Any information or images that you use should be referenced on your one presented slide.
5. Would I be marked down if I read from the script?
No, while we would like you to embody the role of a healthcare professional or medical biophysics expert,
marks will not be given for acting but only on the content of information that you are presenting.
Criteria Excellent Good Satisfactory Not satisfactory
2 marks 1.5 marks 1 mark 0 marks
Analysis, Synthesis & Understanding: Discussion of disease key Mostly accurate discussion of the Generally accurate discussion of The video content does
The video must cover the following contents. symptoms and possible key symptoms and possible the key symptoms and possible not cover the vast
● Discussion of patient symptoms, treatments is comprehensive and treatments, with minor treatments, with minor majority of the required
● Explanation of the underlying biophysics accurate throughout. omissions. Accurate explanation errors/omissions. contents
that is causing the symptoms with Accurate explanation of of the biophysical issues
reference to action potentials the cell biophysical issues underpinning underpinning the disease. Identification of the major
membrane voltages and any other the disease. underlying biophysical issue,
changes, and with some minor errors or
● Possible treatments or cures and how explanation lacking detail.
these affect the patients bioelectricity.
Structure, Organisation & Communication: Information is presented in a Information is presented in a Information is presented in a Information is not
● Information is presented in a clear & logical sequence, and is logical sequence, in a way that is logical sequence, but jargon or presented in a
logical sequence presented in a way that is mostly understandable by a lay language used may not be clear, concise or logical
● Language appropriate for non-expert understandable by a patient or person. A graphic is used with understandable by a patient or manner and cannot be
audience layperson. some value in explaining and layperson. readily understood by
● Effective integration of visuals/figure underlying biophysical principle. the audience.
An accurate and relevant graphic A graphic is used but with limited
is used effectively to explain an value in explaining an underlying No graphic is included,
underlying biophysical principle. biophysical principle. or is
inaccurate/irrelevant.
Creativity Video delivery is highly engaging Delivery is engaging for the Delivery is generally engaging Delivery impacts
● Audience engagement for the target audience, and target audience and there are for the target audience. negatively on audience
● Demonstrates creative or novel elements incorporates a creative or novel creative elements in the engagement (ie. read
element or aspect. presentation. Attempts to include some in monotone voice) No
engaging and/or creative evidence of
A slide, if used, contains relevant aspects. creative/novel aspects.
information using suitable font
sizes and good quality/resolution
diagrams.
1 mark 0.75 marks 0.5 marks 0 marks
Formatting: The video fulfils all of the The video fulfils the majority of The video fulfils some of the The video does not
The video must fulfil the following criteria. formatting criteria. the formatting criteria. formatting criteria. fulfil any of the
● The video is 1 min ± 30 sec in length. formatting criteria.
● The video is submitted in mp4
● The speed of videos has not be artificially
changed
● The recorded sound is clear