Read the text in the main window. ... You will see small windows on the left. Look at ... Estimate this new membrane potential with the Nernst equation below: 11.

Procedure for use of Neurolab program Cell and Mol Biol lab 1. Click on “My Computer” in the upper left portion of your desktop. Then click on “C Drive” and then click on “Nia.htm.” 2. NeuroLab program opens; click on “About…”and look through how accurate the program is, etc. You do not have to look at “Setup,” “Orientation” or “For Instructors.” When done, go back to the home page. 3. Click on “Start NeuroLab” 4. On the left side of your screen (sidebar), click on “Resting Potential” (don’t go into the first lesson but the second) 5. Read the text in the main window. Click and read the link “Nernst Equation” but do not click on “Ena - or EK variations” or “the Membrane tutorial.” 6. After reading about the Nernst equation, click on “Start the Simulation”; then click on OPEN in the next window. 7. You will see small windows on the left. Look at the “Patch Parameters” window. a. the sodium (Na) and potassium (K) channel density is noted; this tells you how many channels are present. The more channels, the faster the membrane potential (Em) changes. Don’t change them now. b. Leakage- a few ions can cross the membrane- or leak across. Ignore this. c. Intracellular Na and K concentrations: record the starting values here (we will change them; to return the starting value, click on the red checkmark that appears after changes are made). d. Ignore Ena, Ek, Eleak.

NOTE: SINGLE CLICK ONLY 8. Under the “Panel and Graph Manager,” click on “Na, K and leak.” A new window appears. 9. Then click on “Run Control” on the “Reset and Run” panel. A new THICK red line appears; this is the Em (membrane potential). It settles on a “stable” value that you can estimate with the Nernst equation. Move the mouse to the thick red line (ignore the thin horizontal red line) and left click; you will see the Em value in the blue bar at the top (it is the y value). WRITE DOWN THIS VALUE HERE:__________ mV (remember to note + or -).

Note your estimate of the Em with the Nernst equation: write down equation with symbols, then plug in proper concentrations to estimate the Em, finally, show your estimate (answer in correct units; R= 1.987 cal/deg mole; Z is charge of the ion, F = 23,062 cal/volt equiv; T =temp in Kelvin (37C +273), Ln is natural log base e= 2.718):

10. Now, change the K concentration outside the cell. If your kidneys fail, K in the blood stream (extracellular K) will shoot up to 80 mM. Change the extracellular K value to this number (from 5 mM) and then hit “Reset and Run” in the “Run Control” Panel. Note that the Em depolarizes to a value of ________ mV.

This depolarization causes the heart muscle cells to contract wildly- the heart begins “ventricular fibrillation (rapid heart beat) that can cause death. It is called hyperkalemia. Estimate this new membrane potential with the Nernst equation below:

11. Next, change extracellular K (and then “reset and run”) to the following values and record the Em: 50 mM 0.5 mM 250 mM

Using Sigmaplot, graph out these four points: put Em (switch to Volts from mV) on the Y axis, and extracellular K on the X axis. Change the axis (which?) to natural log and see if you get a straight line. Print the graph and put it in your lab notebook.

Rearrange the Nernst equation to see what the slope (m) represents. The equation of a straight line of course is y = mx + b (m is slope, b is y intercept). So the Nernst equation can be changed to: Em = Y = (RT/ZF) (ln [K]extracellular or X) - ln [K]inside

M is the slope and equal to the symbols…..

What is the numerical value of m (plug in for symbols)?

Is this the slope you noted in your graph (determine the slope in the semi- log graph as slope is change in Y over the change in X…)?

12. Next change the extracellular Na from140 to 10 mM, and then to 200 mM. Each time, hit Reset and Run to produce the result. Is there any effect on the red line? YES NO (circle one). Explain your answer.

13. Reset all concentration values by clicking on the red arrows. Now we will mimic an action potential in a nerve cell. Remember that during an action potential, sodium channels rapidly open up. So, the membrane changes since now Na is the most permeable ion (use its concentrations to calculate the membrane potential Em). Click on “Na Conductance Only” in the upper left window. Note that the Em goes from about _______ mV to _______ mV. Use the cross hairs to find actual values (left click on the red line, see the Y value in the blue bar above).

In a normal action potential, the Na channels then rapidly close and the cell returns to the resting state where K channels are more open than any other ion.

Using the Nernst equation, estimate the membrane potential when Na channels are open- then compare this value to the value you noted above. Be exact.

14. Now vary the extracellular Na; from 140 to 50 mM. Hit “reset and run” and record the stable Em here: __________ mV. SO NOW CHANGING THE SODIUM CONCENTRATION DOES HAVE AN EFFECT ON THE Em because sodium is more permeable than other ions. 15. Hit “quit” in the P and G manager to end the program.

NOTE: SINGLE CLICK ONLY 8. Under the “Panel and Graph Manager,” click on “Na, K and leak.” A new window appears. 9. Then click on “Run Control” on the “Reset and Run” panel. A new THICK red line appears; this is the Em (membrane potential). It settles on a “stable” value that you can estimate with the Nernst equation. Move the mouse to the thick red line (ignore the thin horizontal red line) and left click; you will see the Em value in the blue bar at the top (it is the y value). WRITE DOWN THIS VALUE HERE:__________ mV (remember to note + or -).

Note your estimate of the Em with the Nernst equation: write down equation with symbols, then plug in proper concentrations to estimate the Em, finally, show your estimate (answer in correct units; R= 1.987 cal/deg mole; Z is charge of the ion, F = 23,062 cal/volt equiv; T =temp in Kelvin (37C +273), Ln is natural log base e= 2.718):

10. Now, change the K concentration outside the cell. If your kidneys fail, K in the blood stream (extracellular K) will shoot up to 80 mM. Change the extracellular K value to this number (from 5 mM) and then hit “Reset and Run” in the “Run Control” Panel. Note that the Em depolarizes to a value of ________ mV.

This depolarization causes the heart muscle cells to contract wildly- the heart begins “ventricular fibrillation (rapid heart beat) that can cause death. It is called hyperkalemia. Estimate this new membrane potential with the Nernst equation below:

11. Next, change extracellular K (and then “reset and run”) to the following values and record the Em: 50 mM 0.5 mM 250 mM

Using Sigmaplot, graph out these four points: put Em (switch to Volts from mV) on the Y axis, and extracellular K on the X axis. Change the axis (which?) to natural log and see if you get a straight line. Print the graph and put it in your lab notebook.

Rearrange the Nernst equation to see what the slope (m) represents. The equation of a straight line of course is y = mx + b (m is slope, b is y intercept). So the Nernst equation can be changed to: Em = Y = (RT/ZF) (ln [K]extracellular or X) - ln [K]inside

M is the slope and equal to the symbols…..

What is the numerical value of m (plug in for symbols)?

Is this the slope you noted in your graph (determine the slope in the semi- log graph as slope is change in Y over the change in X…)?

12. Next change the extracellular Na from140 to 10 mM, and then to 200 mM. Each time, hit Reset and Run to produce the result. Is there any effect on the red line? YES NO (circle one). Explain your answer.

13. Reset all concentration values by clicking on the red arrows. Now we will mimic an action potential in a nerve cell. Remember that during an action potential, sodium channels rapidly open up. So, the membrane changes since now Na is the most permeable ion (use its concentrations to calculate the membrane potential Em). Click on “Na Conductance Only” in the upper left window. Note that the Em goes from about _______ mV to _______ mV. Use the cross hairs to find actual values (left click on the red line, see the Y value in the blue bar above).

In a normal action potential, the Na channels then rapidly close and the cell returns to the resting state where K channels are more open than any other ion.

Using the Nernst equation, estimate the membrane potential when Na channels are open- then compare this value to the value you noted above. Be exact.

14. Now vary the extracellular Na; from 140 to 50 mM. Hit “reset and run” and record the stable Em here: __________ mV. SO NOW CHANGING THE SODIUM CONCENTRATION DOES HAVE AN EFFECT ON THE Em because sodium is more permeable than other ions. 15. Hit “quit” in the P and G manager to end the program.