Besides providing you with the opportunity to familiarize yourself with the basics of action potentials, the experiments you’ll be performing with this simulation have two additional goals.  The first is to conduct a test of the Sodium Hypothesis, which states that it is Na⁺ influx that is responsible for the action potential’s depolarization phase (the upward deflection of the tracing).  The second goal is to conduct a test of the Potassium Hypothesis, which states that it is K⁺ efflux that produces the features of the repolarization phase (the afterpotential and subsequent return to resting V_m). 

Tips and Hints

1.  The exercises you performed when you ran the Membrane Potential simulation provide a good background for this one.  You may want to run the Membrane Potential simulation and familiarize yourself with the results before you run the present simulation and attempt to answer the accompanying questions.

2.  Because of the rather large amount of information that can be displayed on the screen during this simulation, the display can quickly become too cluttered to read easily. This is especially true if you choose to have the simulation display e_Na+, e_K+, and/or e_Cl-.  Therefore, I recommend that you only display equilibrium potentials (especially e_Cl-, which doesn’t change in this simulation) when they’re required to facilitate your understanding of what’s going on or to complete data gathering for a particular exercise.  Regardless or whether you’re displaying equilibrium potentials or not, plan on using the "Clear" button frequently.

3.  You need to record the values for resting V_m, e_Na+, e_K+, e_Cl- generated by the default parameter settings (click Reset if necessary) for use during the Voltage Clamp Experiment simulation that you’ll be working with next. Use the following table to record those results:

Parameter	Value
Vm
eNa+
eK+
eCl-

4.  Your testing of the Sodium and Potassium Hypotheses will be facilitated if you graph the data you obtain from Exercises 1 and 2, below.  If you need assistance in using a spreadsheet to construct graphs, read the accompanying essay “Working With Your Data”.  Also, see hint #5, below.

5.  It will help you understand action potentials better if, before you actually run the simulations, you put yourself in the place of the early investigators of action potentials and try to decide what sort of data you need to support or refute the Sodium and Potassium Hypotheses.

6.  When attempting to answer Questions #2 and #3 below, recall from the Goldman Equation that the flux (J_i) of an ion through a plasma membrane is proportional to the natural logarithm of the ratio of the ion’s concentration (c_i) on either side of the membrane.  That is:

J_i µ ln(c_i,out/c_i,in)

Thus, if you are changing only the external or the internal concentration of some ion, it’s perfectly appropriate to expect that the invariant concentration would ‘drop out’ of calculations and that variation in J_i would be proportional to the natural logarithm of the concentration that’s actually changing.  In other words,

J_i µ ln(c_i,out)

or

 J_i µ ln(1/c_i,in) º - ln(c_i,in)

 

depending on which concentration, c_i,out or c_i,in, is being varied in your experiment.  Keep these expressions in mind when you’re getting ready to graph your data and using the graphs to decide whether your results constitute support for the Sodium or Potassium Hypothesis.

Exercises

1.  Make sure that each of the three Display checkboxes have checks in them.  Conduct a trial run by clicking the Go button.  Observe the general appearance of the action potential’ tracing and familiarize yourself with the information displayed on the screen.  In particular, note that numerical values for V_m at the ‘tip’ of the action potential spike ( = 38 mV) and for V_m at the afterpotential’s maximum ( = -73 mV) are displayed, along with numerical values for e_Na+ (54 mV), e_K+ (-74 mV), and e_Cl- (-65 mV). 

2.  Use the slider or text field to set [Na⁺]_ext to its minimum value of 0.  Conduct a series of runs in which you increase [Na⁺]_ext, in increments of 50 – 100 between each run.  For each run, record the value of e_Na+ and V_m at the 'tip' of the action potential spike.  Continue until you’ve increased [Na⁺]_ext to its maximum value of 1000. 

3.  Use the slider or text field to set [K⁺]_ext to its minimum value of 5.  Click the Go button and note carefully the appearance of the resulting action potential.  Conduct a number of runs in which you increase [Na⁺]_ext, in increments of 50 – 100 between runs, all the way to 1000.  For each run, record the value of e_Na+ and V_m at the 'tip' of the action potential spike.

4.  Use the slider or text field to set [Na⁺]_int to its minimum value of 20.  Conduct a number of runs in which you increase [Na⁺]_int, in increments of 10 between runs, all the way to 100.  For each run, record the value of e_Na+ and V_m at the 'tip' of the action potential spike.

5.  Use the slider or text field to set [K⁺]_int to its minimum value of 200.  Conduct a number of runs in which you increase [K⁺]_int, in increments of 50 between runs, all the way to 500.  For each run, record the value of e_K+ and V_m at the maximum amplitude of the afterpotential.

6.  Reduce the external [Na⁺] to 1 and increase it in increments of 1 and observe the effect on the e_Na+ and the 'behavior' of the action potential tracing.  Note carefully the relative values for e_Na+ and the value of V_m at the ‘tip’ of the action potential’s spike.

Questions

1.  When you increase [K⁺]_ext, what are you doing to the magnitude of the driving force of K⁺ efflux?  When you increase [Na⁺]_int, what are you doing to the magnitude of the driving force for Na⁺ influx?  Which of the two changes has the most dramatic effect on the appearance of the action potential?  Propose an explanation for your observation.

2.  Which, if any, of your results support the Sodium Hypothesis?  (hint:  see Tips and Hints #5, above)  Do any fail to support it?  Defend your responses.  Describe another experimental protocol that would allow you to conduct an additional test of the Sodium Hypothesis. 

3.  Which, if any of your results support the Potassium Hypothesis?  Do any fail to support it?  Defend your responses.  Describe another experimental protocol that would allow you to conduct an additional test of the Potassium Hypothesis. 

4.  Assume that the threshold of this neuron is -50 mV and that Na⁺ channels are 100 % recovered from inactivation by t = 1.4-1.5 ms post-stimulation.  What effect would decreasing [K⁺]_ext have on the ability of the neuron to respond (by generating another action potential) if it received another stimulus at t = 2 ms?

5.  What do the results of Exercise #5 suggest about the driving force for Na⁺ flux?  Can you generalize the results to describe the driving force for transmembrane flux of any given ion?

Data Sheet For Action Potential Simulation

 

Run #	Na+ext	Na+int	K+ext	K+int	eNa+	eK+	eCl-	Resting Vm	Spike Vm,max	Afterpotential Vm,max	Notes