Cystic Fibrosis Transmembrane Conductance Regulator Tutorial
Primary Author: Nerma Jahic
Introduction to Cystic Fibrosis
CF affects approximately 1 in 2000 people in United States and
is the most common lethal genetic disease of Caucasians. This disease causes
certain glands to malfunction. In CF, mucous glands produce thick, sticky
mucus, which interferes with breathing and digestion. Mucus clogs passages
in lungs and airways, causing breathing difficulty, chronic coughing, and
sometimes heart failure. Mucus also blocks ducts in the pancreas
preventing digestive enzymes from reaching the intestines,
and it may also clog the liver and digestive tract. Also, this
thick mucus appears to be an ideal medium for bacteria, which are the
most responsible for deadly symptoms of the disease. Individuals with
severe cases frequently die before the age of 30.
"Cystic Fibrosis Transmembrane Conductance Regulator" is the name for
the gene and the corresponding protein in which the genetic defect for the
cystic fibrosis condition arises. Functionally, the protein is a "chloride
channel" - a protein that resides in cell membranes and permits the
passage of chloride ions across the membrane. Even the normal, fully
functional protein carries the name of the disease, and is called "Cystic
Fibrosis Transport Regulator" (CFTR). Why? Because the location of the
genetic defect was identified in the genome and the corresponding protein
before the function of protein was fully established.
mutations of the CFTR gene result in damage to the CFTR protein, which is
then unable to carry out its function - transmembrane transport
regulation. Cystic fibrosis disease (CF) is a result of the loss of this
To begin to understand how a mutation in the CFTR
gene can disrupt the proper functioning of the CFTR protein and lead to
cystic fibrosis, let us examine the structure of CFTR.
Structure of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
To understand what happens in the body of the individual with cystic fibrosis,
we first need to understand structure of the protein Cystic Fibrosis
Transmembrane Conductance Regulator (CFTR) and the role that the structure
CFTR is an integral membrane protein and is made of a single polypeptide chain of
1480 amino acids. The amino acids are grouped in five domains:
two transmembrane segments, TMD1 and TMD2; two cytoplasmic nucleotide-binding
domains, NBD1 and NBD2; and the regulatory (R) domain. Each of the domains
has a special function when it comes to transport of Cl-ions through the cell
membrane. Therefore mutations in different domains will affect the function of
the protein differently, causing a range of mild to deadly cases of CF.
How do these domains participate in transmembrane transport?
Functional Units (Domains) of CFTR
The two transmembrane domains (TMDs) form the pore through which ions
flow across the membrane. Each TMD contains six membrane-spanning alpha
helices. Many disease-causing mutations occur in TMD1, which are often
associated with milder forms of CF.
The two nucleotide-binding domains (NBDs) gate anion permeability through a pore. The most common
disease-causing mutation in CFTR is a loss of phenylalanine residue at
position 508 in NBD1.
As you can see in the schematic representation below,
The two halves of the protein are connected by the highly charged,
cytoplasmic, regulatory R domain. Today, there are many hypotheses about
what is precisely the function of the R domain, but that question is yet
to be answered.
What happens when there is a mutation in CFTR
CFTR Structure and Function
The loss of only one of 1480 amino acids or its replacement with different
amino acid may make CFTR unable to regulate ionic transport through the
cell membrane. It has been mentioned earlier that the basic defect in
cystic fibrosis is due to abnormalities in the secretions of exocrine
glands. These glands produce protein-containing fluids, which have
different functions. The fluids can have digestive, lubricative or
protective functions. The normal content of this fluid is affected
directly by the ionic, protein and water composition of the epithelial
tissue concerned, which are essential to correct functioning of the
In normal tissue, chloride ions enter the lumen from the extracellular
space through epithelial cells. This creates an increased negative
potential across the epithelial cells, which results in the transport of
sodium ions down the potential gradient into the lumen. Higher
concentration of ions in the lumen causes osmosis of water from the
extracellular space into the lumen.
How is this different from a tissue affected by CF?
The function of a tissue affected by CF
It is known that in CF the luminal side of the affected exocrine gland has
higher negative ionic potential than normal, due to marked decrease in the
permeability of the cell membrane to chloride ions. This was first shown
by an increased concentration of sodium chloride (NaCl) in the sweat of CF
patients. This causes an increased uptake of sodium ions, contributing
further to the negative ionic potential. The increase in chloride ions and
decrease in sodium ions decreases the osmotic movement of water into the
airway, thereby increasing the viscosity of the mucus secretions. This
viscous mucus then becomes an ideal medium for bacteria - the most
responsible for fatality by CF.
The Importance of Molecular Evolution in Relation to Disease
As stated before, the CFTR structure and function will be greatly affected
when a mutation arises, therefore one must look at the molecular aspects
of this protein. It is very important to know the origins of a specific
disease, for only then will one be able to control its manifestations.
Within a gene sequence coding for a specific protein, several mutations have
occurred. These mutations have helped further the evolution of more complex
Therefore, within the specific gene sequences, there will be regions
of Amino Acids which are highly conserved, meaning the comparisons of the
different gene sequences within different species of animals will yield in
a select grouping of different sections of the genes. Some of these
sequences will be alike in the case where they have Amino Acid residues,
other areas will be very different. The areas which have the same
sequences are most likely the most important regions which are responsible
for protein function. This is most likely why different species of
organisms have a conserved region within the protein.
Therefore a mutation within the conserved (alike) regions
will cause deleterious effects, for the integral part of the gene (most
likely the areas which code for the functionality) is altered.
Through comparison with several species of healthy organisms, one may
then determine where the defective mutation is located, and then determine
how to treat the disease.
Finally, after being introduced to elementary concepts of CF and the
relationship between the disease and molecular evolutionary trends,
you are ready for a journey that will enable you to explore many
sides of the CFTR protein using a very powerful tool - The Biology Workbench .
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Tutorial:
- 1) If possible, open a second web browser
window and open the Biology Workbench V.3.2 in it.
Alternatively, we recommend printing the tutorial.
- 2) Go to
- 3) Select Ndjinn- Multiple Database
Search then hit the 'Run' button.
- 4) Scroll down and select the
SWISSPROT database by clicking the small box to the left of the
tag. A check should appear.
- 5) Scroll back up to the object
field and type
CFTR in the object field.
To the right of the field, click the arrow and select "Show All Hits".
Now press the "Search" button.
You will observe that 18 objects
downloaded. The list of those objects represents different sequences of
CFTR protein. Why are those sequences different? They are different
because they "belong" to different species. Even though they are
similar, we will see that on chains of about 1480 amino acids, which build
these sequences, some parts are more alike than the others. Besides that,
with help of the Biology Workbench, we will be able to visualize a
different degree of resemblance that CFTR protein has between different
How to do that? Easy! Just follow the next step.
The Search, Continued
- 6) Highlight your desired entries. IF YOU
ARE ON A PC, HOLD DOWN THE CONTROL KEY WHILE SELECTING THE
tutorial, please highlight the following:
- squac (which is an abbreviation for the taxonomic name of a spiny
- sheep and
- bovin (abbreviation for "bovine"
or a cow).
Of course, you can scroll through the list and pick 5 or 6 other
interesting animals, but we recommend that only when you become more
familiar with the Biology Workbench and you begin some
explorations on your own.
Now click on Show Records
Note that we could choose as many of these sequences as we wish but the
CFTR protein is rather large one and choosing a greater number of
sequences would require more time for the computer to process our request.
For some smaller proteins, such is myoglobin, this number can be exceeded
without having to wait much longer for the results.
The first results are here.
The First Results
A second screen should appear, containing information about CFTR protein
for each of the five chosen species. In addition to information such as
function of protein, subcellular location, tissue specificity, etc., this
screen provides numerous links for those who want to research specific
protein in detail. For those interested in biomedical aspects and
importance of given proteins, there are many links that lead to MEDLINE.
A very interesting web link that is given for cystic fibrosis (under
human CFTR) is http://www.genet.sickkids.on.ca/. This link abounds with
information about CF. We will return to this link after we demonstrate a
couple of aspects of CFTR given in the Biology Workbench.
- 7) On the bottom of the first screen, click on the Import
- 8) On the next screen, click on the box at the
left side of the first five imported objects, then highlight in the
operations menu CLUSTALW- Multiple Sequence Alignment and then hit
- 9) The next window will present many choices for alignment
parameters. Simply select Submit to submit the alignment job with
the default parameters.
As one may see after scrolling down the page, a list of the five
large sequences are shown. Several of the letters (which correspond to
different amino acids) are the same from top to bottom (the five
sequences). These are the conserved regions that were mentioned
- 10) Now, hit Import Alignments
and you will see a little box to the left of the set of the aligned
sequences. Click this box, and then within the box above, find
DRAWGRAM- Draw Rooted Phylogenetic Tree from Alignment.
Hit Run. Once again, use the default parameters and hit
Submit. Now see what happens:
Personalized Phylogenetic Trees
You will see a schematic representation that we could call a small,
"personalized phylogenetic tree." While observing this tree, bear in mind
that it conveys quite a large amount of information about evolution
of these few species. We can see how closely related one species is to
another. While looking at this tree, try to answer some questions:
How long ago did two species have a common ancestor?
We can see that "branches" of this tree differ in length.
Another possibility is to view a second (more easily readable) phylogenetic
tree and do the following:
- 11) Hit the "Return" button at the end of the page, and the
Biology Workbench will take you back to the set of aligned
sequences that you clicked on to view the first tree. Instead of clicking
on DRAWGRAM, click on DRAWTREE-Draw Unrooted Phylogenetic Tree
from Alignment and hit "Run." Using the default parameters, hit
View this tree, and compare it to the one that was viewed previously. Now
you may answer the following questions:
- How closely related are the organisms to one another, as well as
its relation to the center point?
- What do you think the centermost
- What is the difference between a Rooted and Unrooted
Now, go back to the original set by once again hitting Return.
As done previously in this tutorial, the sequences were aligned so that
the phylogenetic analyses could be performed. Now, it is possible to
view this alignment and display the conserved regions.
Let us now view the alignment itself.
Viewing the Alignment
12) Click on the square to the left of the alignment, highlight
BOXSHADE, and select Run.
13) On the following screen again submit the job with default parameters
by selecting submit.
And here is the payoff!
The final result is a display of a color-coded alignment, which shows
the regions that are "highly conserved" across various species. Each
letter in "protein strings" represents one of 20 amino acids that are
found in biological organisms. Those amino acids are also known as
"essential amino acids." For those unfamiliar with abbreviations and
single letter designations for amino acids we suggest printing The Symbols
for Amino Acids.
In our color-coded alignment, different colors represent different
degrees of conservation of certain regions accross species.
The green regions are completely
conserved across all the species, the yellow regions are highly conserved, the
light blue regions are somewhat
and the unshaded regions are highly
variable from one species to another.
Conserved regions are believed to contain a specific amino acid
composition that is essential for main function of CFTR protein- transport
of chloride ions through cell membrane. Therefore the conserved regions
tell us what is necessary for CFTR to be CFTR.
On the other hand, we have certain mutations in protein structure
had happened long time ago. Some of those mutations were allowed to
survive because they have not impaired the function of the protein. In
some cases they have even enhanced it. That is the reason why today we
have "variable regions." The degree of difference between one species and
another tells us the evolutionary distance.
So what can we learn from our example?
What can we learn from our example?
Take a look at your aligned sequences. If you examine bovine, sheep and
spiny dogfish CFTRs what you can conclude? Does bovine have CFTR that is
more similar to sheep CFTR or to spiny dogfish CFTR? What would you
However, we have to have in mind that we are looking at normal, functional
CFTR. What happens in a case of cystic fibrosis? If you want, you can
compare normal and CF CFTR or continue exploration of the CFTR protein.
There are many more ways to research the CFTR with The Biology Workbench
than you might suspect.
Let's see a couple more.
Compare normal and CF CFTR.
Look how long the CFTR chains are. Each one of them contains
about 1480 amino acids. The human CFTR contains exactly 1480 amino acids.
Pretty long string, isn't it? Imagine all amino acids having positions
from 1 to 1480.
Suppose now that we make just a small, tiny change and lose amino acid
that is position 508. That amino acid is phenylalanine, marked F in our
colorful alignment. Among so many amino acids you would hardly notice
that position 508 is now empty. It would not look like such a big deal. But
In reality, a person with the loss of phenylalanine at position 508 has
one of the most severe cases of cystic fibrosis. Individuals with severe
cases frequently die before the age of 30.
Let's see a couple more.
Detecting Transmembrane Segments
- 14) Now press the Return button at the bottom of the screen.
will take you to the screen where we have previously highlighted BOXSHADE,
but this time you want to highlight TMAP- Prediction of Transmembrane
segments and hit Run.
The next screen will give us data about number of TM segments for CFTR,
start and end point of TM segments, and graphical presentation of those
segments for different species.
Where I can find more information on Cystic Fibrosis?
Finding More Information
Now we can go back and "Follow
The Link" given in section - VIEW THE RECORDS.
Chromosome 7 Project takes us to
Project and genetic aspects of CF as well to general
There is an interesting link at the bottom of the screen -
Genome Database (GDB) and can be used
when searching for a specific gene.
Clicking on major Cystic Fibrosis Mutation
Database rectangle leads us to a page that is dedicated more
exclusively to Cystic Fibrosis and CFTR:
Table gives us overview of nucleotic changes and their consequences
on protein level.
- CFTR Gene
Sequence link gives connection between DNA bases and amino acids.
variation of common Cystic Fibrosis mutations is link to resources
that concentrate more on relevant statistics. The sub-links that might
be worth exploring are:
- CF-WEB link - many different levels of
information about CF and CFTR.
Check these links: