Cftr Protein As The Cause Of Cystic Fibrosis Biology Essay

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Cystic fibrosis (CF) is characterized by a defect in the chloride ion channels of the epithelial cells, resulting in a buildup of mucus in airways, damage to the lungs and destruction of the pancreas (Goodman and Percy 2005; Karp 2010). It is caused by a mutation in the CF transmembrane conductance regulator (CFTR) protein, a chloride channel in the apical membrane of epithelial cells (Hamai, Keyserman et al. 2009). CFTR is an ATP binding cassette (ABC) transporter, and has 2 membrane domains, each of which are comprised of six membrane spanning components, most of which are α-helices, and following each domain is a nuclear binding domain, NBD1 and NBD2, which cause conformational changes for the membrane domains (Hanrahan and Wioland 2004; Wang, Wu et al. 2010). The two membrane domains are connected through the regulatory domain (R-domain), which had many sites for phosphorylation for both Protein Kinase A and C (Hanrahan and Wioland 2004). The activation of the CFTR protein depends on the phosphorylation of the R-domain, as well as the ATP hydrolysis of NBD1 followed by the binding of AMP-PNP to NBD2 (Hanrahan and Wioland 2004). In some CF causing mutations, it is possible that both or one NBD is blocked, which causes inactivation of the CFTR protein (Hanrahan and Wioland 2004).

The role of the activated CFTR is to act as a c-AMP mediated chlorine channel that transports chlorine out of the epithelial cells, and maintains water balance (Proesmans, Vermeulen et al. 2008; Karp 2010). However, when CFTR transports chlorine out of the cell, due to a high permeability for bicarbonate ions, an exchange occurs where chlorine leaves the lumen and bicarbonate enters (Ishiguro, Steward et al. 2009). Therefore, in cystic fibrosis where CFTR does not work properly, there is an excess of chlorine inside the epithelial cells and an excess of extracellular bicarbonate ions. Additionally, CFTR normally suppresses activity of sodium ion channels, thus if the CFTR protein is mutated and not properly functional, there is an increase of extracellular sodium, as compared to normal concentrations (Goodman and Percy 2005; Karp 2010).

The mutant CFTR can be grouped into five different mutant classes. Class one mutants are usually due to a deletion, frameshift or non-sense mutation, which prevents CFTR from even being produced. Class two mutants are characterized by abnormal intracellular CFTR protein trafficking, while class three mutants are due to defects in the regulation of CFTR as a chlorine channel (Rubenstein 2005). These three mutations cause more severe CF than class four and five mutants, which are caused by reduced function of the chlorine channel and decreased expression from normal protein function, respectively (Rubenstein 2005).

The second class of mutant CFTRs is the most common, accounting for 70% of CF cases (Rubenstein 2005). The normal CFTR gene has a phenylalanine group at position 508, and a deletion of this group (from NBD1) causes CF (Grove, Rosser et al. 2009). Without F508, NBD1 does not fold properly and, therefore the CFTR protein is rejected in quality control (Grove, Rosser et al. 2009). In this process, the GT enzyme recognizes that CFTR is not folded properly and re-adds a glucose and as a result, CFTR again enters the quality control cycle (Karp 2010). However, because ΔF508 is deleted from the protein, it will not be able to re-fold and bypass quality control, and thus cannot go to the plasma membrane and will be ubiquinated (Grove, Rosser et al. 2009). It is the entire process that is the reason why the CFTR protein is not present in the plasma membrane in most CF case, and why ion balance in a CF patient is irregular.

In less common cases, CFTR will reach the membrane but will have adverse effects within the plasma membrane (Hamai, Keyserman et al. 2009). It is suggested that it preferentially localizes within lipid rafts, areas of high concentrated cholesterol and sphingolipids (Hamai, Keyserman et al. 2009). Furthermore, it has been found that the ΔF508 mutation in CFTR results in an increase of sphingolipids in the epithelial cell membrane, which in turn will interfere with signalling proteins, however an unclear connection between increased sphingolipids and damaged human lung epithelial cells as a result of the mutated CFTR remains (Hamai, Keyserman et al. 2009).

There are many possibilities for treatment for CF, but two areas relate most closely to CFTR, which are pharmaceuticals that affect ion concentrations across the membrane to substitute for the defective CFTR, as well as gene therapy (Proesmans, Vermeulen et al. 2008). Possible pharmacological agents include Amiloride, which blocks the overactive ENaC to reduce the high extracellular sodium ion concentration, MOLI 1901, which stimulates chlorine channels other than CFTR or drugs such as Denasol, which uses synthetic nucleotides to activate purinergic receptors to stimulate calcium-activated chlorine channels (Proesmans, Vermeulen et al. 2008) . These examples of current treatment, among many other drugs, function to replace or regulate the function of CFTR, when CFTR itself is not present or properly functional. Gene therapy is another potential therapy for CF, and has good potential because only a small amount of functional CFTR is needed to restore chlorine balance (Proesmans, Vermeulen et al. 2008). However, thus far gene therapy has been unsuccessful as there is no vector that has effectively integrated the normal CFTR gene, however research in this area is ingoing, and using lentiviruses as a potential vector is being explored (Proesmans, Vermeulen et al. 2008).


Annotated References

Goodman, B. E. and W. H. Percy (2005). "CFTR in cystic fibrosis and cholera: from membrane transport to clinical practice." Advan. Physiol. Edu. 29(2): 75-82.

The clinical perspective of CF is the main subject of this article, and it discusses the effects of the mutated CFTR protein on the lungs and the pancreas. It emphasizes the destruction that CF can potentially have on the pancreas as well as the lungs, and provides a few ideas for treatment, such as oral enzyme supplements.

Grove, D. E., M. F. N. Rosser, et al. (2009). "Mechanisms for Rescue of Correctable Folding Defects in CFTR{Delta}F508." Mol. Biol. Cell 20(18): 4059-4069.

The purpose of correct protein folding in regards to the CFTR protein, and how an F508 deletion causes the inability for the protein to properly fold is explained in this paper. It addresses quality control, and how the class two mutations rarely bypass quality control to reach the membrane.

Hamai, H., F. Keyserman, et al. (2009). "Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signalling." J. Lipid Res. 50(6): 1101-1108.

Hamai discusses the effect that a mutated CFTR protein has in the plasma membrane of epithelial cells. The main conclusion from this study was that mutated CFTR proteins cause an increase in the synthesis of sphingolipids, which will interfere with some of the cell's signalling mechanisms.

Hanrahan, J. W. and M.-A. Wioland (2004). "Revisiting Cystic Fibrosis Transmembrane Conductance Regulator Structure and Function." Proc Am Thorac Soc 1(1): 17-21.

The makeup of the CFTR protein is discussed and how each different part interacts is one of the main focuses of this article. To focus is generally on the interactions of the NBDs and the other parts of the CFTR protein, and it is suggested that some CF-casing mutations might directly block one or both NBD gates.

Ishiguro, H., M. C. Steward, et al. (2009). "CFTR Functions as a Bicarbonate Channel in Pancreatic Duct Cells." J. Gen. Physiol. 133(3): 315-326.

This paper discusses the role of CFTR not only as a chloride channel, but also as a channel for bicarbonate to enter the cell. It is the high permeability of a normally functioning CFTR protein that allows bicarbonate to enter the cell.

Karp, G. (2010). Cell and Molecular Biology: Concepts and Experiments, Wiley & Sons.

Karp discusses the CFTR protein as the cause of CF, introducing the subject by explaining the clinical consequences of CF, and briefly how CFTR causes these consequences. CFTR's role as a chlorine channel, as well as being a bicarbonate conductor and a sodium channel suppressor is explained, and treatments are briefly mentioned. Overall, Karp provides the basic knowledge of CFTR of CF.

Proesmans, M., F. Vermeulen, et al. (2008). "What's new in cystic fibrosis? From treating symptoms to correction of the basic defect." European Journal of Pediatrics 167(8): 839.

This article discusses many different aspects of the CFTR gene and CF, however it provides a very good explanation regarding some of the more popular treatments for CF, and how they interact at a cellular level. The future of CF research is discussed, with the emphasis being on gene therapy as potentially the most effective treatment for CF.

Rubenstein, R. C. (2005). "Novel, mechanism-based therapies for cystic fibrosis." Current Opinion in Pediatrics 17(3): 385-392.

The genetics of the CFTR mutation are the main points this article addresses. There are 5 different types of CFTR mutations that can cause CF, and to treat CF, repairing the CFTR protein is not enough; there are many other functions that this protein serves which also must be restored.

Wang, W., J. Wu, et al. (2010). "ATP-independent CFTR channel gating and allosteric modulation by phosphorylation." Proceedings of the National Academy of Sciences: -.

The article discusses how the CFTR protein acts as a channel, including how the different parts of the protein such as the NBD and membrane domains interact. Conformational changes in the NDBs are mentioned.

Personal Reflection

While writing this paper I found one of the most difficult aspects to be going through the literature, and finding the most relevant information, as there is so much information on the CFTR protein and on CF, it was sometimes hard to pick out the most important and also accurate information. The best strategy I found to address this problem was to use information that I consistently found repeated in more than one paper, or newer information that had built on the research of others.

I found it very interesting to learn exactly the mechanisms of how one defective protein causes such huge problems on a clinical level, and how many possible interactions at a cellular level can cause a disease such as CF.