Modeling Ion Movement
In this lab we will explore the importance of ion balance in neurotransmission. In part one you will model how ions move in and out of a neuron due to channels opening. Then in part two you will use a computer simulation to look at how ion distributions across the cell membrane (plasma membrane) make an action potential possible.
You will make a hypothesis that talks about how ion balance is important for neuron transmission. Remember that transmission (communication from one neuron to a another) can only happen when an action potential is triggered. Here are some questions to consider when framing your hypothesis. Make a hypothesis that integrates these questions.
⦁ Can an action potential be triggered without ion flow occurring?
⦁ Can an action potential occur if there is not the correct concentrations of ions on the outside and the inside of the cell?
Now that you have a hypothesis we will test the hypotheses with some modeling! In part one we are using physical models to look at how channels opening allow ion flow down their concentration gradients. In part two we will use a computer model to ask how important the precise ionic concentrations are to be able to trigger action potentials.
Lab 3 – Part 1:
Understanding ion flow:
Grab 1 clear plastic cups, 1 paper cups, and a pin. Put an inch of water in the clear plastic cup, fill the paper cup with water. Put the paper cup inside the clear plastic cup. Add 5-10 drops of food coloring the water inside the paper cup. Now, lift the paper cup up, take the pin and puncture the side of the paper cup. Now put it back into the clear plastic cup. Describe what happens.
Insulation:
Using two magnets and a paper towel, allow the magnets to connect through the paper towel. Repeat but this time fold the paper towel. Can the magnets still connect through the folded paper towel. Continue folding the paper towel until the magnets cannot connect. What have you demonstrated here?
Answer these questions in your lab report in the methods section as you are describing them. Remember to state the methods in a narrative form and not a numbered list.
⦁ What does the water inside the paper cup represent based on our lectures?
⦁ What kind of channel does the pin hole represent (be specific!!)?
⦁ What ion does the food coloring represent?
⦁ What does the paper towel represent?
⦁ What do the magnets represent?
Lab 3 – Part 2:
The knowledge you gained from the food coloring should help build your hypothesis about how a cell’s membrane potential is controlled by changes in the ion concentration. Form a hypothesis about what will happen as you decrease Na+ concentrations outside the cell. Remember this hypothesis as you will need it for the lab!
Next go to this Simulator for Neural Networks and Action Potentials (SNNAP) https://nba.uth.tmc.edu/neuroscience/s1/labs/actpot/hhsimu.html
by the University of Texas Health Science Center at Houston. You will land on a page showing the one below.
The blue line Vm represents the membrane potential that is calculated using this equation.
In order to understand anything about this equation you will need to know what the terms in the equation mean. As stated above, the Vm is the membrane potential, Cm is the membrane capacitance (capacitance is the ability of a membrane to store a charge and is determined by physical properties of the membrane), Iinj is the injected current, gNa is the Na+ conductance, gK is the K+ conductance, and gl is the leakage conductance. ENa and EK are the equilibrium potentials of sodium (Na) and potassium (K) respectively. There are also a few variables that model activation of ion channels. Recall that ion channels are what gives the membrane permeability to the ions. Ions themselves will not pass-through membranes. There had to be a hole in the cup to let the ions (dye molecules) out. The conductance of the Na+ channel is governed by an activation variable m and an inactivation variable h, gNa = gNamax m3h and the conductance of the K+ channel is governed by a single activation variable n, gK = gKmax n4
Causing and Action Potential with Channel Opening.
Now that we understand the terms, we can start manipulating some of them. The default settings here are set to use an electrical impulse (Current Injection) to trigger an action potential (blue line). The orange and green lines model the increase in permeability of sodium and potassium ions due to the opening of sodium and potassium channels due to an electrical impulse. Notice that there is a term for Current Injection. This is the stimulus that will trigger the opening of the ion channels in the membrane because here we are modeling voltage gated ion channels. When you set this Current Injection to zero, there will be no permeability of the membrane to ions (all channels are closed).
Answer these questions in your results section.
⦁ What happens to the membrane potential when the Current Injection is zero?
⦁ Alter the Current injection to be 5 nA/cm2. Do you see an action potential being fired now?
⦁ Step up the Current Injection from 5 nA/cm2 one step at a time leaving all other parameters set to their default. At what current do you see the action potential fire?
Answer these questions in your results and discussion section.
⦁ Knowing that Current Injection causes ion channels to open, explain what has happened when the current reaches the level needed to cause the action potential. Make sure to mention channels opening and increased permeability of ions.
Altering Ion Concentrations
We know from the Nernst and Goldman equations mentioned in the beginning of this lab that the equilibrium potential for ions can be simplified (over simplified) to a ratio of the concentration of an ion outside a cell to that on the inside of the cell [ion]outside/ [ion]inside. Mathematically this means that as the concentration of the ions outside a neuron is lowered so will the equilibrium potential of that ion. Reducing the concentration of sodium ions outside side of a neuron would mimic what happens in cases of hyponatremia (low sodium and too much water) that we saw in “The Agony of Ecstasy MDMA and the Kidney” paper.
Notice that you can also change Equilibrium potential of both sodium and potassium. In this exercise we will manipulate the equilibrium potential of sodium to model hyponatremia. When we reduce the equilibrium potential of sodium, we are effectively decreasing the amount of sodium outside the cell and breaking down the concentration gradient of sodium that exists across the neuron membrane. Recall that ions will move from areas of high concentration to areas of low concentration (flowing down the concentration gradient).
The default settings on our simulator models a standard sodium gradient that you would see under normal conditions. This results in an Equilibrium potential of sodium of 55 mV. To model hyponatremia, change this to 0 mV while keeping all other parameters the same.
Answer these questions in your results section.
⦁ Where you able to trigger an action potential while the equilibrium potential of sodium was zero? Note that the action potential trace is still in a dotted blue line so you can see what it would look like.
⦁ Note the membrane potential at the peak (apex of blue line) when the Equilibrium potential of sodium is 0 mV and check that it matches what is on the chart below. Determine the membrane potential at the peak for four more points of sodium equilibrium potential levels anywhere between zero and 55 and fill in the chart below.
Sodium Equilibrium Potential (mV) Membrane Potential (mV)
0 -41.5
For your graphics section.
⦁ Make a scatter plot of Membrane Potential vs. Sodium Equilibrium Potential. Since Sodium Equilibrium Potential is the independent variable it should go on the x-axis (Abscissa) and since the Membrane Potential is the dependent variable it should go on the y-axis (Ordinate). Add a trendline and determine the coefficient of determination (R2). For more information on the R2 and what it can tell us ⦁ look here. What trend do you notice? How can you tell if it is significant?
In your discussion section answer the following.
⦁ Now let’s imagine a scenario like the one in the JAMA article about ““The Agony of Ecstasy MDMA …” where the sodium outside the neuron is really low and thus causing the equilibrium potential of sodium to be low. Based on your plot above do you think that action potentials (depolarization of the membrane) will be more difficult in hyponatremia?
⦁ How might this change in action potentials affect consciousness?
⦁ How might this change in action potentials affect your heart’s ability to beat?
⦁ Why does taking ecstasy sometimes result in this?
Introduction: Introduce the lab and your hypothesis. Give pertinent background information as needed that explains what you are doing.
Methods : Talk about what you did for the lab to collect the data and/or information as well as the analyses you did.
Results : Talk about the data you collected and the results of the analysis without analyzing it.
Discussion : Analyze the data in your results and relate that to your hypothesis. The discussion section will also be where you talk about how to improve your research and why this type of study matters.
Summary of what was found and why it matters. Make sure to answer the questions given in the lab report for this section.
Clear statement of whether the hypothesis was supported or rejected and why.
Statement(s) of what the strengths of the experimental approach were.
Statement(s) of what the weaknesses of the experimental approach were including possible sources of error or bias.
Graphics :
Give a completed chart of your values from part 2 and an XY scatter plot of these values.
Appropriate labels are provided for each graphic. This include chart titles and axis labels.
