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Home , Action potential, Depolarization, Repolarization, Resting potential, Threshold potential

A Message to Teachers

Thank you for using our tools to help your students visualize the molecular world! The lesson/activity guide that accompanies the Kit is intended to help you consider different ways in which you may use these materials. It is not the intent of the Center for BioMolecular Modeling to require you to work through the entire lesson from start to finish (although you may do so if you wish). We do encourage (and perhaps insist on) your modification of these lessons and activities to meet the learning objectives of your specific students or to accommodate the physical limitations of the environment in which you teach. Enjoy!

Part I: Modeling the

Introduction:

All living cells, including cells, maintain a difference in the concentration of across their membranes. These ions play an essential role in neuronal signaling. Ions are unequally distributed between the interior of the and the surrounding fluid. A small excess of negatively charged ions on the inside of the and a small excess of positively charged ions on the outside of the cell membrane results in a negatively charged cell interior relative to the outside environment. This difference in electrical charge across the membrane can be measured with a voltmeter and ranges from -60 to -80 mV (millivolts) when the is not sending a signal. The at rest is referred to as the neuron’s resting potential. For the purpose of this lab activity, we will consider a neuron with a resting potential of -70 mV. A membrane that exhibits a membrane potential is said to be polarized.

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Set up the membrane of the of a neuron according to the diagram to the right.

The permeability characteristics of a neuron’s plasma membrane are, in part, Outside determined by specific channels. Channels in the nerve cell membrane that transport negatively charged ions are relatively few in number or are closed and not displayed in the model. Inside

1a. Label the membrane, the - gated channel, the leak channel, the voltage-gated and the sodium-potassium pump in the diagram. Outside

Table 1. Average Concentrations Inside and Outside of Mammalian

Intracellular Concentration Extracellular Concentration Ion (mM) (mM) Potassium (K+) 140 5 Sodium (Na+) 15 150 (Cl-) 10 120 Large anions (A-), such as 100 Negligible some , inside the cell

Potassium ions (K+) represented by the square purple models and sodium ions (Na+) represented as round blue models are the key players in establishing the resting potential. According to the table above, in most neurons, the concentration of Na+ is higher outside the cell while the concentration of K+ is higher inside the cell. Distribute the sodium and potassium ions to illustrate this observation in the model you have constructed.

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1b. In a resting neuron, what positive ion is most abundant outside of the plasma membrane? (Na+)

1c. The K+ gradient dictates that potassium ions will flow in what direction? (K+ flows out of the cell)

1d. What happens to the resting potential of the cell when K+ ions move? (As K+ ions move out of the cell, the inside of the cell becomes more negative relative to the outside of the cell.)

1e. What direction does the concentration gradient dictate the Na+ ions flow? (Na+ ions will flow into the cell)

1f. Carefully examine the sodium-potassium pump. What is the exchange ratio of Na+:K+ ions of the sodium potassium pump? (HINT: Focus on the shapes in the pump.) (For every three sodium ions pumped out of the cell two potassium ions are pumped in)

1g. What molecule is necessary for the sodium-potassium pump to work against the chemical gradient? (ATP)

1h. What factors impact establishing the resting membrane potential?

1. The Na+ and K+ gradients are maintained by the sodium-potassium pump. The energy of ATP hydrolysis is used to actively transport three Na+ out of the cell for every two K+ into the cell.

2. The chemical gradient favors a net flow of the of K+ through potassium channels (often called leak channels) out of the cell.

3. Very few Na+ channels are open as compared to K+ channels. Because K+ ions more readily cross the membrane due to potassium leak channels and Na+ and other ions can’t readily cross the membrane, K+ outflow leads to a relative negative charge inside the cell as compared to the outside of the cell.

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Optional Activity: Comparing the Voltage-Gated to the Voltage-Gated Potassium Channel

1i. How does the voltage-gated potassium channel distinguish sodium ions from potassium ions? (Sodium ions travel through their channel in the hydrated state. The potassium is designed so that potassium ions are not hydrated when they travel through the channel.)

1j. What structural differences can you observe in the channel pore of these voltage-gated channels? (The sodium channel is “wider” than the potassium channel to accommodate the hydrated sodium ion while the potassium channel is narrower to accommodate only the potassium ion. The voltage-gated potassium channel is “lined” with oxygen atoms that are precisely spaced to accommodate a potassium ion and not a sodium ion.)

Part II: Modeling the

Set up an axonal membrane as shown in the diagram below and to the left. Remember to set up your model so that there are more extracellular Na+ ions than intracellular Na+ ions and more intracellular K+ ions than extracellular K+ ions. You will also need to periodically refer to the graph shown below and to the right as you work your way through the modeling process:

The Action Potential

Changes in membrane potential in a local area of a neuron's membrane result from changes in membrane permeability.

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Next, you will simulate the mechanism that produces an action potential in the axon of the neuron. Follow the steps below to model this action potential.

Step 1 - Resting state: The voltage-gated sodium and potassium channels are closed. Set your voltmeter at -70 mV.

Step 2 - : A opens the voltage-gated sodium channels and Na+ follows its concentration gradient into the neuron. The influx of Na+ causes a depolarization across the cell membrane. An action potential will be triggered if the depolarization reaches threshold (often between -40 and -55 mV). Set the voltmeter at threshold. For the purposes of this activity, we will consider the to be -50 mV.

Step 3 - Rising phase of the action potential: Depolarization opens most of the voltage-gated sodium channels, while the voltage-gated potassium channels remain closed. Open the voltage-gated sodium channels and move a few more Na+ into the neuron. Depolarization continues until the inside of the membrane is positive with respect to the outside (usually +30 mV). Set the voltmeter at the peak value of the action potential. When the patch of neuron membrane is generating an action potential and its voltage-gated sodium channels are open, the neuron cannot respond to another stimulus no matter how strong.

Step 4 - Falling phase of the action potential: Voltage-gated sodium channels become inactivated, blocking Na+ inflow. Voltage-gated potassium channels slowly open permitting K+ to follow its concentration gradient out of the cell causing the voltage across the membrane to fall. Position the voltage-gated sodium channels to their closed and inactive position. Open the voltage-gated potassium channel and move the K+ ions out of the neuron. Demonstrate a fall in voltage due to this ion movement on the voltmeter. Only a stonger than normal stimulus can reopen the voltage-gated sodium channels at this time.

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Step 5 - Hyperpolarization: The voltage-gated sodium channels begin to reset back to their original position. Move the voltage-gated sodium channels to their closed but able to be activated position. The voltage-gated potassium channels are still open causing the voltage to undershoot the resting potential. Set the voltmeter to dip below - 70 mV.

Step 6 - : Repolarization restores resting electrical conditions but does NOT restore resting ionic conditions. The ion redistribution is accomplished by the sodium-potassium pump.

Step 7 - Re-establish the ion distribution:

7.1 7.2 & 7.3 7.4 7.5 & 7.6 7.7

7.1: Bind three three intracellular sodium ions to the appropriate spots in the .

7.2: Bring the ATP in close proximity to the pump.

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7.3: Sodium ion binding stimulates phosphorylation of the pump protein by ATP. In other words, a phosphate group is added to the sodium-potassium pump from the ATP molecule. (You will not be able to demonstrate this step with the model).

7.4: Phosphorylation causes a change in the shape of the protein. You can demonstrate this by “swinging” the sides of the protein so that it opens to the outside of the cell.

7.5: The shape change reduces the protein’s binding affinity for sodium ions and increases the binding affinity for potassium ions. Remove the sodium ions from the protein and deposit them outside the cell and bind two potassium ions to the appropriate spots in the protein.

7.6: Potassium ion binding triggers the release of the phosphate group from the protein. (Again, you will not be able to demonstrate this step with the model).

7.7: Loss of the phosphate group results in the restoration of the protein’s original shape which then releases the potassium ions. Swing the sides of the protein back so that they open to the inside of the cell and deposit the potassium ions.

7.8: Repeat this process one more time.

2b. What value is being used for the resting potential in your model neuron? (-70mV)

2c. What “factors” may cause a change in the voltage established across the membrane this neuron? (A change in voltage may occur as a result of a stimulus such as heat, light, pressure, chemicals, a change in body position or a change in pressure.)

2d. Of what significance is a rise in membrane potential to -55mV to the neuron? Describe what protein and how it is affected by this change in voltage. (This particular neuron has reached threshold potential at -55 mV meaning that depolarization becomes self generating (the all-or-none response). The voltage-gated sodium channels open allowing sodium ions to follow into the cell.)

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2e. What is meant by depolarization? (Depolarization is a reduction in membrane potential – the inside of the membrane becomes less negative (moves closer to zero) than at resting potential.)

2f. Color the area on the graph to the right where depolarization has occurred.

2g. Sketch the axon membrane that has been depolarized. Be sure to include all channels and ions in their proper locations and positions.

Outside

Inside

Outside

2h. What events occur at the peak of the action potential? (The voltage-gated sodium channels are inactivated and the volta ge-gated potassium channels are opened.)

2i. Why is it important that another action potential cannot be generated during the rising phase of an action potential? (Because the voltage-gated sodium channels are open in a particular section of the membrane, an action potential cannot be generated there. The inward current that depolarizes the axon ahead of the action potential cannot produce another action potential behind it ensuring neuron transmission occurs in one direction.)

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2j. Why does the voltage across the neuronal membrane fall? (When the voltage-gated potassium channels open, positively charged potassium ions leave the neuron causing the membrane potential to fall.)

2k. Color the area on the graph to the right where repolarization has occurred.

2l. Sketch the axon membrane that has been repolarized. Be sure to include all channels and ions in their proper locations and positions.

2m. Why does the voltage “undershoot”? (The voltage-gated potassium channel gates close more slowly. Excessive K+ efflux results in hyperpolarization.)

2n. Color the area on the graph to the right where hyperpolarization has occurred.

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2o. Fill in the following to compare voltage fluctuations and ion concentration changes that occur during an action potential.

Relative Relative Relative Relative Action Potential Voltage Intracellular Na+ Extracellular Na+ Intracellular K+ Extracellular K+ Step (mV) Concentration Concentration Concentration Concentration

Resting -70 Low High High Low

Threshold -55 Increasing Decreasing High Low

Peak +30 High Low High Low Depolarization

Falling to Repolarization High Low Decreasing Increasing resting

Hyperpolarization ~-75 High Low Low High

2p. Why is the sodium-potassium pump necessary after an action potential has been generated? (While repolarization restores the electrical potential, the resting potential ion distribution has not been restored. The concentration of sodium and potassium ions is the opposite of what it should be at resting potential. That is to say that the concentration of potassium ions is greater outside than inside the cell and the concentration of sodium ions is greater inside than outside the cell. The sodium-potassium pump is necessary to reset the ion distribution to resting potential conditions.)

3q. What would happen to a neuron if threshold potential is not reached? (The neuron would not “fire” an action potential.)

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