Na+/K+ ATPase and Electrochemistry
Part I: Introduction
Need Cn3D for structure viewing
http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3dinstall.shtml
Purpose: To show how electrochemistry applies to biology, how to use Nernst equation
Introduction to electrochemistry –
Before we begin, it will be useful for you to review the chapter on electrochemistry in
your textbook – particularly the section on the Nernst equation. It will also be useful for you to
know a few key terms.
Using your textbook or other sources, define the following terms in your own words:
Ion, voltage, electrical potential, membrane potential
A brief introduction is presented here:
A membrane potential exists when there is an imbalance in the number of positive and negative
charges on either side of a cell membrane. This imbalance represents disequilibrium, and we
know that when a system is not at equilibrium, there is free energy available to do work.
Recall that ΔG = -RT ln Keq and when the system is at equilibrium, Keq = 1, therefore ΔG = 0.
The term potential really means that there is potential energy stored in this system - just like a
spring that has been stretched. Energy is required to stretch the spring and to keep the spring
from recoiling. If you let go of the spring, it will immediately recoil to its relaxed (or
equilibrium) state. The same is true of the potential energy stored across membranes. In order to
establish and maintain this potential, there must be an energy input. This energy input ultimately
comes from ATP. Here’s how:
Membrane potentials exist in cells because there are more negative charges inside the cell than
outside – this is referred to as an electrochemical gradient. This happens because positive
charges are actively pumped out of the cell by a membrane protein called the Na+/K+ ATPase.
This protein uses energy from ATP to pump three sodium ions (Na+) out of the cell for every two
potassium ions (K+) it pumps in. So the net result is that the outside of the cell becomes more
positive than the inside. The chemical energy stored in the phosphate bonds of ATP is converted
into potential energy in the form of the membrane potential.
How does ATP provide this energy? It does so by transferring a phosphate group to the ATPase
protein. The ATPase normally binds 3 sodium ions on the inside of the cell. But when the
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phosphate group is transferred from ATP, the protein undergoes a conformational change
causing it to release these ions into the extracellular space. The protein then binds 2 potassium
ions from outside the cell causing the protein to again change shape and release the potassium
ions on the inside of the cell. If that was confusing, try watching this video:
http://highered.mcgrawhill.com/sites/0072495855/student_view0/chapter2/animation__how_the_sodium_potassium_pu
mp_works.html
Part II: Structure of the Na+/K+ ATPase
Next, let’s look at the structure of the Na+/K+ ATPase in a more precise manner. Threedimensional structures of proteins can be determined at very high resolution using a method
called X-ray crystallography. The details of this method won’t be discussed here, but it is a very
common and useful way to study the structure of proteins. When researchers determine the
structure of a protein, they deposit that structure in a centralized database which can be accessed
through the Nation Center for Biotechnology Information (NCBI) website. Follow this link to
check out the structure of the Na+/K+ ATPase.
http://www.ncbi.nlm.nih.gov/sites/entrez?db=Protein&itool=toolbar
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Change the dropdown menu to “Structure” and type “sodium potassium atpase” into the search
box as shown above.
Click on “38BE”
Click on “Structure View in Cn3D” > “Open”
This is the 3-D structure of the Na+/K+ ATPase. Note that you can rotate the structure around
and look at it from different angles. The sites on the protein that say “Rb” are actually the K+
binding sites (For technical reasons, Rb+ was used in this study as a replacement for K+). On the
sequence/alignment viewer, A, B, G, C, etc correspond to different domains of the protein. The
letters to the right are amino acids – the building blocks of proteins. On the A domain, amino
acid number 327 interacts with potassium ions. This amino acid is a glutamic acid (one-letter
abbreviation is E). The side chain structure of glutamic acid is shown below:
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O
O
-
R
Glutamic acid side chain (R=backbone structure)
Electrostatically speaking, why would this particular amino acid be useful in a K+ binding site?
Highlight this amino acid on the Sequence/Alignment Viewer by finding position 327 and
clicking on it. When the cursor is hovering over this position, you should see “(PDB 327)” in the
lower left corner.
Now the amino acid should be highlighted on the 3D picture. See if you can find it. It is part of
the blue spiral that has turned yellow. Don’t worry if you can’t find it, it’s a little hard to pick
up. Check it out on the picture below. The cursor is pointing to amino acid 327.
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The potassium binding site of the Na+/K+ ATPase
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Part III: Membrane Potential and the Nernst Equation
It is possible to calculate the magnitude of an electrochemical potential using the Nernst
equation:
E = -2.3(RT/zF) log10[Ci]/[Ce]
In the context of biological systems, this equation relates electrochemical potential (E) to the
concentrations of an ion inside the cell [Ci], and the concentration of that ion outside the cell
[Co]. Note that the other two variables in this equation are temperature (T) and charge of the ion
(z). R and F are both constants. For our purposes, all of our calculations will use a temperature
of 37°C (or 298K) and a charge of +1. Therefore, the quantity -2.3(RT/zF) will equal -60 mV.
This will simplify the calculations. Making this substitution, we get:
E = -60 log10[Ci]/[Co]
Note that if an ion is more concentrated inside the cell than outside, the potential will negative,
and if the reverse is true it will be positive.
The other equation that will be useful to us relates maximum free energy to electrical potential:
ΔG = -nFE
Here n = number of ions (in moles) and F is again a constant.
Next we’re going to use Microsoft Excel to graphically visualize what happens to membrane
potential when ion concentrations change. For this exercise, we are interested in what happens
to the potassium membrane potential as extracellular potassium changes. Let’s assume that the
potassium concentration inside the cell stays constant at 120 mM.
Graph the potential (in mV) vs. potassium concentration outside the cell.
The first step in Excel is to make a list of potassium concentrations ranging from 0.5 to 15 in
increments of 0.5.
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©2011 University of Illinois Board of Trustees • http://islcs.ncsa.illinois.edu/copyright
Now in column B, tell Excel to calculate the potassium membrane potential (EK+) using the
Nernst equation for each concentration of extracellular potassium. Start in cell B4 and then drag
that cell down so that Excel will perform the same operation on each cell in column B. Assume
that the intracellular potassium concentration ([K]i), is constant at 120 mM. Now plot
extracellular potassium concentration ([K]o) on the x-axis versus potassium membrane potential
on the y-axis. If you need help using Excel to perform these tasks, ask your teacher to guide you.
Part IV: Questions
Looking at the graph, what happens to the membrane potential as [K]o increases?
This is referred to as depolarization, since the electrochemical gradient of K+ across the
membrane decreases. What can we say about the equilibrium of this system? As [K]o increases,
does the system approach equilibrium or get farther from it? How do you know?
What about the potential energy of the system?
Cardiomyocytes are the muscle cells of the heart. Coordinated contraction of these cells allows
the heart to pump blood throughout the body. The average potassium potential for
cardiomyocytes is -85 mV. Looking at your graph, estimate the average extracellular potassium
concentration (this is equivalent to the blood potassium level).
When potassium levels in the blood become high, this is called hyperkalemia. Hyperkalemia is a
dangerous medical condition because it can cause irregular contractions of the heart called
arrhythmias, which can sometimes be fatal. In fact, the lethal component of lethal injections in
the United States is potassium chloride. It is used to drastically elevate blood potassium levels
causing the heart muscle to stop functioning.
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Heart arrhythmias begin to occur when blood potassium levels reach about 8 mM. Again
looking at your graph, what would the membrane potential be?
An interesting side note: A drug called digitalis has been used for centuries in the treatment of
heart failure. It is isolated from the plant Digitalis purpurea (seen below).
Digitalis purpurea (the common foxglove plant)
www.landscapedia.info/plant.php?plantID=15303
Basic structure of digitalis
http://commons.wikimedia.org/wiki/File:Digitalis_glycosides.png
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Digitalis works by inhibiting the Na+/K+ ATPase pump. If given in the proper dose, this is
actually therapeutic, but if too much digitalis is ingested, the heart will stop. The foxglove plant
is therefore highly toxic. One of the side effects of digitalis intoxication is xanthopsia – a visual
disturbance that causes the world to look yellow, similar to looking through a yellow filter.
Other visual side effects include glare and colored haloes. Interestingly, some have argued that
Vincent Van Gogh may have experienced these side effects after being treated with digitalis and
this may account for some stylistic aspects of his art – including his propensity to use yellow
colors and the colored haloes seen in his paintings (see below).
http://www.psych.ucalgary.ca/pace/va-lab/AVDE-Website/VanGogh.html
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