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Structure and Function of the CFTR Chloride Channel

Meena Subramaniam Cluster 8 COSMOS 2009

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Meena Subramaniam COSMOS 2009, Cluster 8 July 30, 2009

Structure and Function of the CFTR Chloride Channel Abstract: This research paper focused on learning more about the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR), with the help of previously published journal articles. CFTR is a chloride channel in the body which, when mutated, causes the disease cystic fibrosis. It was learned that CFTR is an ABC transporter, meaning that it uses ATP to open and close. Phosphorylation of the R domain allows for two molecules of ATP to bind to the nucleotide-binding domains (NBDs) of CFTR, and a signal is transmitted to allow for the opening of the channel. ATP binding to the NBDs is crucial to the opening of the channel, as strain from the dimer formed forces the channel into an open conformation. Channel closure is determined by the hydrolysis of one of the ATP molecules which binds to the NBDs, and once ATP becomes ADP (thereby releasing a phosphate group), the channel begins to close. When hydrolysis occurs, the channel no longer has to accommodate the strain caused by dimerization, and thus the channel closes. Mutations in the "signature sequence" of the NBDs (such as a substitution or deletion of an amino acid) eliminate ATP binding to the channel, thus eliminating channel opening and closure entirely. Scientists are very interested in how ATP binds to the protein channel, and how mutations in the NBDs disrupt the ATP binding process. Analogs of ATP have been experimented with, though most do not bind as well to the NBDs as ATP does. ATP analogs have the potential to be incorporated into drugs which could practically cure certain

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mutations of CFTR such as Phe508 by promoting normal channel gating. In addition to this, it was found that the metal cation Cadmium 2+ activates channel gating in mutated G551D CFTR. This may be the basis for research on metal cations which are more readily ingested by the body, such as Calcium 2+ or Zinc 2+. Keywords: ABC Transporter, dimerization, Phe508 mutation, G551D, phosphorylation

Introduction: The cystic fibrosis transmembrane conductance regulator (CFTR) is a channel which transports chloride ions across a cell membrane. It is particularly important to the study of the disease cystic fibrosis. Cystic fibrosis is the result of a mutation which makes the CFTR chloride channel ineffective or unable to open. When chloride cannot cross the cell membrane, anion flow is decreased, causing a buildup of mucus in the lungs. In order to create more effective drugs to treat cystic fibrosis, the structure and mechanisms of the CFTR protein channel as well as the effects of the common mutations must be analyzed. CFTR is quite unique in its nature, due to its function. It is classified as ATP Binding Cassette transporter, or an ABC transporter. The name refers to the fact that the channel uses ATP (Adenosine triphosphate) binding to open and close normally. Interestingly, CFTR is one of the only ABC transporters that behaves like an ion channel, suggesting that CFTR evolved from other ABC transporters to function as an ion channel. Because it is so different from other chloride channels, CFTR is difficult to understand and represent. In fact, a full crystal structure has yet to be mapped out for CFTR. Despite not knowing the overall organization of this protein channel, scientists have made progress in identifying how ATP binding allows for CFTR channel

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opening and closure, as well as how mutations in the "signature sequences" disrupt the channel gating process.

Structure of CFTR: The CFTR chloride channel is made up of five domains. These include two transmembrane domains (TMDs), that, as their name suggests, reside in the membranes of cells, two nucleotide binding domains (NBDs) that are crucial to ATP binding, and a regulatory domain. 1

Figure 1: Rough structure of CFTR portrayed in two ways 2

The TMDs each consist of six alpha helices, which are connected to each other. The two TMDS (TMD1 and TMD2) are thought to be symmetrical to each other, as they are in bacterial chloride channels, although scientists are unsure of this assertion. Though most of the alpha helices of the TMDs remain unexplored, the sixth helix in TMD1 is of particular interest to

1 Hwang, T. (2009).Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.

Journal of Physiology, 587, 2151-2161. 2 http://www.unc.edu/~awrs/images/Fig2.jpg

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scientists. The Argenine and Lysine amino acids that reside in the sixth helix are positively charged, and may be the reason that anions are attracted to enter the pore and cross the channel. While CFTR is mainly responsible for the transport of chloride, it is not specific to that anion. Other halide ions can also pass through the channel with relative ease. CFTR's low selectivity is highly contrasting to the selectivity of potassium channels in the body. This can be partially attributed to the configuration of the helices which create the pore 3 . Studying the NBDs in CFTR is more complex and crucial than studying the TMDs because NBDs are essentially responsible for channel opening and closure through ATP binding. Like bacterial TMDs, they were once thought to be identical and symmetrical to each other; studies now show that they are in fact not identical. This contributes largely to the way the CFTR channel opens. Two molecules of ATP are involved in opening the chloride channel. The first ATP binds to the Walker A and Walker B motifs of NBD1. The Walker A and Walker B motifs basically contain hydrophobic, aromatic, and hydrophilic groups to which ATP can bind favorably. More specifically, the Lysine amino acid in the Walker A motif interacts with a phosphate group of ATP, and aromatic sidechains of the Walker B motif allow for the stacking of aromatic groups as well as delocalization. The better the aromatic stacking, the higher the affinity the ATP molecule has to the NBD. The first ATP molecule also binds to the LSHGH "signature sequence" of NBD2 when the channel dimerizes. The second ATP molecule binds to the Walker A and Walker B motifs of NBD2 as well as the LSGGQ signature sequence of NBD1 when the channel dimerizes. Basic differences in the signature sequences of NBD1 and NBD2 cause NBD2 to be more catalytic in the process of channel opening. This means that mutations in NBD2 cause far more changes than those in NBD1. Part of the reason for this is that the ATP

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Gadsby, D. C., Vergani, P., & Csanady, L. (2006). The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature, 440, 477-483.

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molecule which binds to NBD2 acts as the trigger to open the channel, and hydrolysis of the same ATP molecule triggers the closing of the channel.

Figure 2: Process of ATP binding, channel opening and closure 4

Interaction between the NBDs is evident when dimerization is observed. This is because there is significant hydrogen bonding between the NBDs, as well as a high affinity between the and phosphates of ATP with the dimerized complex. ATP binding has become almost a whole field of study to scientists looking to learn more about CFTR channel gating. Many ATP analogs have been experimented with to see if they bind more efficiently and effectively to the NBDs. Some of these analogs include pyrophosphate, whose and phosphates may bind with stronger affinity to the NBDs, as well as P-ATP, whose hydrophobic groups may lead to better binding. Still, a more effective analog than ATP is yet to be discovered and tested.

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http://www.ucl.ac.uk/npp/myimages/PV3.jpg

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Not a lot is known about the R domain of the CFTR channel. At the most basic level, the R domain connects NBD1 to TMD2, and may possibly be required to transmit signals between the two domains. Scientists are fairly certain that phosphorylation of the R domain is required before ATP can bind and the channel can open. Phosphorylation is the process by which a phosphate group is added onto an enzyme or protein channel with the purpose of activating the channel. What is interesting about CFTR is that it is the only ABC transporter which contains a phosphorylation site. Furthermore, the R domain has to be phosphorylated by the very specific protein kinase, or PKA. These specifications make CFTR a very complex channel that is often quite difficult to study.

Process of Channel Opening and Closure: Phosphorylation is essentially the first step to opening the CFTR channel. Next, one ATP molecule has to bind to the NBD1 binding site, where it is attracted to the Walker A and Walker B motifs, as previously explained. The second ATP molecule is then attracted to the Walker A and Walker B motifs of NBD2. (Scientific experiments show that the concentration of ATP as well as PKA from phosphorylation somewhat determine the rate of channel opening. In high concentrations of both molecules, the channel will open more rapidly. However, in lower concentrations, more time will be required to fully open the channel. ) When both ATP molecules are bound securely, they also have interactions with the LSHGH and LSGGQ signature sequences of NBD1 and NBD2, respectively. In this situation, a head-to-tail dimer is formed, where strong hydrogen bonding brings NBD1 and NBD2 together, with the ATP molecules between them. Dimerization causes strain in the molecule, and this strain then forces the TMDs apart, creating the open conformation of the channel. After a certain period of time,

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the ATP molecule bound to NBD2 hydrolyzes, and a phosphate group detaches itself from ATP, leaving ADP. Since ADP cannot bind successfully to NBD2, it leaves the system, and the dimer of NBD1 and NBD2 is broken apart. The rate of channel closure is dependent on several factors. Some include temperature and the concentrations of Magnesium ion and ATP in the environment.

Mutations of CFTR: The two most common mutations of CFTR are the Phe508 mutation and the G551D mutation. The Phe508 mutation is basically a mutation where a phenylalanine amino acid is deleted from the signature sequence of NBD1. The Phe508 mutation was initially thought to cause conformational differences in the protein channel. However, recent studies show that CFTR with the F508 mutation does not have irregular folding. In fact, the stability of the F508 mutation CFTR channel is equivalent to that of the normal CFTR channel, as shown by free energy calculations 5 . The physical changes of CFTR are actually attributed to the fact that mutated CFTR protein is less soluble in water than normal CFTR channels. Scientists have discovered that the F508 mutation induces a chemical change in the CFTR protein which causes it to be a poor receptor of signals. This is especially important since the F508 mutation occurs near the NBD1-TMD1 junction. Thus, the phenylalanine amino acid probably acts as an important transmitter of a signal. Without the phenylalanine, signal transmission is most likely altered, and the channel gating changes. The conclusion that the mutation causes a chemical problem rather than a physical one is vital to creating an effective drug to battle cystic fibrosis.

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Lewis, H.A, et. al. (2005).Impact of the F508 mutation in the first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure. The Journal of Biological Chemistry, 280(Jan. 14), 1346-1353.

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Figure 3: Phe508 mutation 6

The G551D mutation is less common in cystic fibrosis patients, but definitely as significant as the Phe508 mutation. G551D occurs in the signature sequence of NBD2. This mutation is different from Phe508, since it does not even allow ATP to bind to NBD1 and NBD2. This lack of ATP binding causes the channel gating to be extremely slow and inefficient. More experiments have been done concerning G551D; the main conclusion is that even though ATP cannot bind to the channel, ATP analogs do a fairly good job of improving channel gating. In fact, when other seemingly harmless mutations are introduced to the protein, ATP analogs actually bind to the channel with high affinity 7 . As mentioned earlier, a lot of experimentation has been conducted using P-ATP as well as pyrophosphate. In addition to this, studies show that

http://genome.gsc.riken.go.jp/hgmis/posters/chromosome/Gifs/Nbd1.jpg Bompadre, S. G., Li, M., & Hwang, T. (2008). Mechanism of G551D-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Potentiation by a High Affinity ATP Analog. Journal of Biological Chemistry, 283, 53645369.

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G551D CFTR channels can actually open and close with abilities similar to when they are exposed to the Cadmium 2+ ion 8 .

Concluding Remarks: As elaborated above, significant research has been done on CFTR in the past few years. However, in order to develop a more effective drug to use against Phe508 and G551D mutations, CFTR's unique qualities need to be explored in more depth. In order to create a drug to cancel the effects of the Phe508 mutation, the chemical signals transmitted through NBD1 and TMD1 must be researched. Once this has been achieved, a drug can be developed to aid the signal transmission, or possibly to send a signal of its own through the channel. These developments will hopefully lead to effective channel gating. As for the G551D mutation, further analysis of ATP analogs will give scientists more information on how to create a drug that will bind with high affinity to the NBDs. As mentioned earlier, the hydrophobic, hydrophilic, and aromatic groups of the ATP binding sites of the NBDs can be used as targets for a possible drug which can bind to a G551D CFTR channel in place of ATP. Since Cadmium 2+ is not the most ideal ion for activation of the channel (the body rejects it and does not handle it favorably), experiments with a more suitable ion such as Calcium 2+ or Zinc 2+ can be conducted, with hopes of finding an ion that might be able to control and improve channel gating. Scientists are still a long way from understanding the complete structure of the CFTR protein channel, and finding a more effective drug to combat cystic fibrosis. However, as we

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Wang, X., Bompadre, S. G., Li, M., & Hwang, T. (2009). Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel. Journal of General Physiology, 133, 69-77.

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continue to learn more about the physical as well as chemical qualities of CFTR, we can inch closer to creating a drug which can induce CFTR channel opening and closing.

Acknowledgments: I would like to thank Dr. Toby Allen, Dr. Dean Tantillo, and Dr. Annaliese Franz for all of their help in supporting my project and giving me suggestions. I would also like to thank Danny Delgado for his support in helping me organize this paper.

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Structure and Function of the CFTR Chloride Channel