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.

Certain 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 essential function.

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 plays.

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 structure?

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 tissue.

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 organisms.

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:
The Search

  • 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 Protein Tools.
  • 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
    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 were 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 species.

How to do that? Easy! Just follow the next step.

The Search, Continued


For this tutorial, please highlight the following:

  1. human,
  2. mouse,
  3. squac (which is an abbreviation for the taxonomic name of a spiny dogfish)
  4. sheep and
  5. 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 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 Sequences button.
  • 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 "Run".
  • 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 previously.

  • 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. Why?

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 Submit.

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 region is?
  • What is the difference between a Rooted and Unrooted phylogeny?

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 conserved, 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 expect? Why? 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 it is.

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. This 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.

Clicking on Chromosome 7 Project takes us to Human Genome Project and genetic aspects of CF as well to general Genetics web page and Science News.
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:

  1. Mutation Table gives us overview of nucleotic changes and their consequences on protein level.
  2. CFTR Gene Sequence link gives connection between DNA bases and amino acids.
  3. Population 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:
  4. CF-WEB link - many different levels of information about CF and CFTR.

Check these links:

This page created and maintained by Kristian N. Engelsen.
E-mail any questions or comments.