BACK TO DIAGRAM CFTR REVIEW PAGE
The chain of events which set CFTR activation in motion are what we will concentrate on first, however it should be remembered that many of the details are still being worked out. For example, it has yet to be determined whether the NBD domains are opened by the process of ATP hydrolylsis itself or the loss of the hydrolysis products from the NBDs afterwards, among many other controversies. We'll start by picturing ourselves as the ion channel CFTR sitting in the membrane of an epithelial cell in the lung. And we are situated in the membrane of the cell in such a way as to be facing the lumen of the lung (i.e. the hollow tubules). The extracellular part of our channel is situated outside of the cell and therefore projecting into the lumen (where all the dirt, mucus, and bacteria is), while the intracellular 70% of us is inside the safety and comforts of the cell, sort of leading the good life, waiting for the proper commands from our cell to tell us when to open our pore and let chloride ions out of the cell via this pore, out into the lumen of the lung. The rest of us is sitting within the cell membrane itself. This transmembrane portion of the ion channel is the part which forms the pore. Under normal circumstances, CFTR is probably "off", meaning there is no chloride traveling thru the pore. Since we are therefore in the deactivated state, our R-domain has no phosphate modifications added onto it yet, which also means that our NBD domains are not binding any ATPs, even though there's plenty of ATP floating around inside the cell. Perhaps our R-domain is directly inhibiting our NBD1 from picking any of it up. Recent experiments indicate that NBD1 and the R-domain bind each other directly. At any rate, our NBDs haven't bound any of ATP yet even though they will shortly once we get the appropriate signal from the cell. It is also likely that the N-terminal "tail" of CFTR is bound to another protein in the membrane called syntaxin 1A and that this protein is helping to keep us in the "off" position as well by not allowing the R-domain to activate NBD1.
Next, the person who owns the lung cell we are residing in encounters an irritant in the environment which has managed to make its way down into the lung from the outside world. This irritant could be anything from a harmless speck of dust or a pollen grain to a potentially lethal virus or bacteria. It doesn't really matter because the immune system operates under the assumption that an over-reaction to a foreign invader is better than no reaction at all, which is also the reason many of us suffer from allergies, but that's another story. These specialized immune cells in the lungs recognize the invader as foreign and dutifully send out a chemical signal into the blood stream telling the rest of the body that the bad guys have made their way into the lungs and the body had better do something about it, and fast. Sneezing would be nice. So would an increase in mucus in the lungs as well as faster swaying cilia hairs which would all help to push irritants out of the lungs. However, if the body is going to increase the amount of mucus in the lung, it had better also increase the amount of water which keeps the mucus moist and free-flowing, otherwise this strategy could backfire and the lung could end up becoming a trap for bacteria; bacteria which is always on the lookout for a nice place to live and raise a family of its own.
So, the signal that was first initiated by the immune system cell's encounter with the dust particle is now in the blood and on its way to all the cells in the lung, and it turns out that this signal is a chemical messenger called a hormone. The hormone sent out in this case is called VIP (for vasoactive intestinal peptide). It travels thru the blood all the way to the cell we are at, in the lung, and contacts a protein receptor also residing in the membrane, on the outside part of the membrane of the same cell we are in. This receptor is specialized for recognizing the VIP hormone and is called the Beta-Adrenregic Receptor. This protein receptor is in the part of the cell membrane far away from us, however. In fact, it is situated on the opposite side of the cell (called the "basolateral" side) which means it is in contact with the bloodstream, where all the hormones, and not to mention food are. But the large distance separating us from the receptor (i.e. the interior cytoplasm of the cell) doesn't matter a whole heck of a lot because this receptor has a trick up its sleeve for relaying to us, the chloride channel, that it has recently bound the VIP hormone message that is in the bloodstream. And it does this even though we are way on the other side of the cell. The receptor relays the message to us by directly activating another protein (called a G-protein). G-proteins are the "on and off" switches of the sub-cellular molecular world, and this particular G-protein switch is able to in turn activate yet another protein (which performs its job as an enzyme) called adenyl cyclase. This particular enzyme "factory" has the ability to churn out lots of a second messenger inside the cell called cyclic AMP (often abbreviated as cAMP). cAMP is a small molecule compared to a protein, which means it is free to move very quickly, and it therefore crosses from one side of the cell to the other in no time at all. This cAMP is able to "diffuse" inside the cell over to near where we are where it encounters yet another protein enzyme that happens to be near us, waiting for the cAMP signal. It is called PKA (for protein kinase A). PKA binds the cAMP and this binding changes PKA into an active enzyme which is then able to add small chemical groups called "phosphates" onto other proteins such as the R-domain of CFTR. Some proteins become down-regulated by this kind of modification, but in our case, it will help to activate us and open our pore so chloride can eventually flow out of the cell and into the lumen of the lungs. Then, sodium will follow the chloride by going around the cell from the bloodstream into the lumen. Which will have the combined effect of moving water from the bloodstream into the lung and keep the mucus nice and hydrated.
The R-domain is unusual because no other protein in the ABC Transporter Family to which CFTR belongs has one. At first, back in the early 1990s when CFTR was just beginning to be understood, it was thought by some that the R-domain simply formed a kind of "plug" which filled up, or blocked, CFTR's pore from the inside and this kept chloride from leaking out whenever the R-domain was without its phosphate modifications. This was fittingly called the "ball and chain" model and was based on what was known about voltage-gated ion channels. And when the enzyme PKA added phosphates onto the R-domain, the R-domain was then thought to move out of the way of the pore and let chloride pass on thru it. We now know that this view was too simple. Like a lot of things in life. What is currently believed is that the structure of the R-domain is divided up into two separate halves, a part which is unstructured until it is phosphorylated, and another part which stays in a more defined structure even without phosphates. The R-domain probably inhibits channel opening by interacting with the NBDs directly, although this physical type of interaction is hard to prove, experimentally. The N-terminal tail of CFTR could be controlling the R-domain's access to the NBD1. Remember at the beginning that the N-terminal tail was bound to syntaxin 1A? It's been theorized that syntaxin 1A is "lured away" from CFTR during activation by fusion of an "intracellular vesicle" to the cell membrane. If these vesicles contain a protein called syb, which is classified as a v-SNARE, syb could divert syntaxin 1A away from the N-terminal domain by binding to it, which would then allow the N-tail of CFTR to "swoop down" and bind the phosphorylated R-domain, causing it in turn to bind to NBD1 at a stimulatory site, thereby stabilizing NBD1.
What all this means to us, however is that the R-domain acts as an inhibitor of the ion channel when it is not phosphorylated by PKA. Which ties in nicely with what was originally believed back in the early 1990s. It just does so in a way more complicated than simply acting as a "block". And here's where it gets even more interesting. The R-domain also appears to act as a stimulator of CFTR when PKA adds the phosphates onto it. It is unusual for a single protein domain to have more than one job. Most protein domains have either one function or another. Not two separate ones. Perhaps the R-domain is really two separate domains. Kind of like two domains within a single domain. Its as we alluded to earlier, CFTR's R-domain turns out to be special. We should note before continuing on that the most recent evidence seems to suggest that, when all is said and done, the R-domain appears to play more of an inhibitory role than a stimulatory one.
The cell has now told us by way of PKA phosphorylation of our R-domain to commence the opening of the channel pore. For me this always conjures up images of medieval castles with drawbridges and dragons and damsels in distress whose fates often depend upon the gatekeeper's swiftness and surety of action. So by this analogy, the drawbridge would probably be equivalent to a section of the pore near the intracellular side of the membrane. Possibly a site that would normally form part of the lining of the pore when the channel is open, but when closed this small section of the pore blocks off the pathway for chloride permeation thru the channel by using a mechanism reminiscent to raising of a drawbridge. This could occur by the induction of "steric hindrance" imposed by the side chains of the amino acids of the drawbridge section of the pore as opposed to a raising of some kind of "chemical free energy barrier". In other words, it this section of the pore "gets in the way", physically, like a bouncer outside Studio 54. How could all of this happen based what we know currently about how proteins and protein systems function? There is some evidence that this part of the pore opens up as a result of the following sequence of events.
First NBD1 is released from the inhibitory effects of the R-domain, thereby picking up ATP, and then changing shape enough for the R-domain to next recognize a second (different) site on the NBD1, which has the effect of causing the NBD1 domain to then hydrolyze its bound ATP. ATP hydrolysis has been shown by many previous experiments to induce enough of a shape change in protein domains (some of them eerily similar to NBD1 in sequence) so as to put it into an entirely new conformation that can have far reaching effects not only for the protein undergoing the conformational change itself, but also for other proteins that may interact with that particular protein when it is in the new conformation. (Note: this also appears to be what sets off signal transduction events that can often lead to some kinds of cancer when not controlled properly by other kinds of cellular gatekeepers. These particular signal transduction proteins are concerned with helping the cell grow and divide and are referred to as "oncoproteins", but are not involved here with CFTR.) It now looks like we may be able to regard CFTR as some sort of a miniature self-contained signal transduction pathway all its own and self-contained within a single large CFTR protein. In this scenario, the domains would be pictured as interacting with each other as would distinct proteins in a typical signal transduction pathway. CFTR probably has enough "molecular landscape" for all of these interactions to occur. In addition, CFTR has been shown conclusively to be composed of several individual domains. Not to mention the fact that CFTR weighs in at over 160 kilodaltons (1480 amino acids), which is pretty big even for an ion channel.
So as we have seen, NBD1 provides the "gatekeeper" function of CFTR. Which means that it is NBD1 that ultimately controls when the drawbridge section of the pore is open following binding and subsequent hydrolysis of ATP at NBD1. And the way NBD1 opens the pore is by binding to this section of the pore directly. It might be possible to envision the R-domain as helping the process of channel activation along simply by getting out of the way of NBD1 and allowing NBD1 to move more in the direction of the closed drawbridge part of the pore. It may even be possible that, when the channel is closed, the R-domain is binding to the exact same place (the "drawbridge") where the NBD1 will bind once activated. So recapping: all of this "structural amplification" which took place causing the pore to open originally came about courtesy of the signal generated by the cell's VIP hormone receptor. The receptor for VIP (like the immune cells) turn out to be analogous to the "lookouts" for the castle. The drawbridge is now down (CFTR's pore is open), and the damsel can get away from the dragon (chloride can flow out of the cell and into the lumen of the lung, helping to hydrate the mucus; which helps keep the mucus moving; which helps move the irritating dust speck back out of the lung from where it came; which was the point of all this. Geez.).
By everyday human time-scales, ion channels don't stay open very long. Thousandths of a second, more or less. But averaged over time, CFTR is open just over half the time (up until it is deactivated). As soon as NBD1 was able to hydrolyze its bound ATP, the process of activation (pore opening) took only a few microseconds (which is about 3 orders of magnitude less time than the channel will remain open during bursts). At this point, it should be mentioned that many investigators are not quite so sure that ATP hydrolysis at NBD1 is as tightly coupled to channel gating as has been described here. It may also be that the second NBD domain, NBD2, is playing a larger role than anticipated by some of the simpler models. It isn't know yet.
Here are a few more details you can skip if you want to: Recent experiments have revealed that before opening, CFTR may have two different closed states which have been designated as C1 and C2. It's possible that once NBD1 binds ATP, CFTR is able to cycle between the two closed states in a reversible manner until ATP is hydrolyzed at NBD1. Once in C2, it is easier for CFTR to go all the way to the open channel configuration. Also, CFTR may be able to first open briefly before ATP hydrolysis, and stay open longer after ATP is hydrolyzed at NBD1. These so-called "substates" appear more often to investigators when CFTR is studied in a purified fashion than when it is studied in the actual cell membrane along with everything else that normally occurs there in the cell membrane. And as if it couldn't get any more complicated, it appears that exactly which open state CFTR is in could even depend on the extent to which the R-domain is phosphorylated by PKA. R-domains with more phosphates on them seem to favor longer channel open times.
Once fully open, the channel will remain that way slightly over half the time its conductance is measured. All ion channels periodically shut off briefly while in the open state. While open, over 10 chloride ions are able to flow thru the pore before CFTR will let an inadvertent sodium or potassium cation thru; and the channel pore will probably accommodate two chloride ions at any one time. Up to a million chloride ions will travel thru the pore for every second it is open. Some researchers theorize that both NBD1 and NBD2 are working together during the process of channel activation and deactivation, and kind of "help" each other along this way. It still isn't known for sure if this is true because it is very difficult or perhaps even impossible at this time to design an experiment to find out. One of the things that is known, however, is that the process of channel opening seems to depend on temperature somewhat more than the process of channel closing does. This implies that a different sequence of events is occurring at the NBD domains depending on whether the channel is in the process of opening or closing, and that the two processes, opening and closing, are therefore very distinct processes, and could perhaps be considered separate.
What is the "driving force" that makes chloride ions want to leave the lung cell and travel thru CFTR's pore into the lumen? It is mostly due to the negative charge on the interior of the cell's membrane. Since chloride is an anion, meaning it is negatively charged, it wants to escape the negatively charged interior of the cell and go towards the positive exterior. It is possible to actually observe these ions travel thru a single CFTR molecule using a highly sensitive instrument called a "patch clamp" because these chloride ions, when they move thru the pore, create a current which can be measured; in some ways like an electric current when it moves thru a wire.
It has been shown pretty convincingly that CFTR is able to regulate the gating of other types of ion channels in the same lung cell, including a sodium ion channel (called ENaC) and another type of chloride channel; one that lets chloride out faster than CFTR does (called ORCC). One possibility for how this regulation of other ion channels could be accomplished is by way of the NBD domains of CFTR. It appears that they may form dimers with each other, which implies that they could interact this way with other proteins as well. Perhaps these other ion channels CFTR is known to regulate are able to get close enough to CFTR to receive their instructions by binding directly to the same "PDZ domain-containing proteins" that are known to bind CFTR at its C-terminal tail. This would have the effect of bringing CFTR and these other types of channels closer together than they otherwise might be in the cell membrane. Close enough for CFTR to activate them by using its own activated NBD domains. Most of the amino acids composing the intracellular loops are hydrophilic, however not all of the loops are of the same overall length. Some of the larger loops are predicted to have secondary structures like alpha helices. It is possible that the intracellular loops of CFTR participate in gating as well as activation of other ion channels.
A question that has been asked many times in the years since CFTR was shown to be an ion channel in 1992 is: what part of CFTR forms the pore? Many investigators have tried to answer that question by using the method of "mutagenesis", where specific amino acids are changed in the protein and the subsequent effects on ion conduction by the pore are determined using activity assays such as the patch clamp method described above. If a particular amino acid residue (like lysine, for example) turns out to be important for ion conduction thru the pore, when it is changed to a different amino acid, a lower conductance of chloride ions thru CFTR should be seen. Interpretation of the results is where problems often occur when using mutagenesis to study ion channels. Was the change in ion conductance after mutation of the lysine because the lysine was a binding site for chloride? Or was the change in ion conductance due to an overall structural change in CFTR due to removal of the lysine and that change just happened to affect the pore structure, albeit indirectly? Interpretation is important in mutagenesis studies. And yet mutagenesis is a very powerful technique when the results are interpreted the right way. .
The path the chloride ion takes thru CFTR from the interior of the cell to the exterior of the cell should define for us where the pore is. The model which so far has described CFTR the best is one based on a channel with 3 binding sites for chloride inside the pore. It appears that there are at least one, and possibly two, spaces, or vestibules, within the pore. The one nearer to the inside of the cell has been well documented and appears to have a positively charged amino acid specifically for binding anions like chloride. What causes the chloride to travel thru CFTR's pore?
First, a chloride ion inside the cell is electrostatically attracted to the surface of CFTR near the opening of the pore by using positively charged amino acids in the intracellular loops of the protein. This serves to increase the "effective concentration" of the chloride ions near the pore. At this point, the chloride ion is still surrounded by several hydration layers of water, so the ion is large. Too large to go into the pore. In order to shed some of the water molecules in the outer hydration shells, the CFTR protein must begin to make contacts with the chloride ion to make up for the loss of the waters that the ion must suffer. It probably overcomes this energetically unfavorable situation by using the backbone carbonyl groups of the transmembrane helices lining the pore as well as hydrophilic amino acid side chains like serine or threonine. The pore also needs to make sure that chloride and not some other type of ions make it thru. It therefore needs to employ some kind of "selectivity", probably by using a selectivity filter. Often in ion channels, this type of selection takes place in the narrowest part of the pore. Some evidence suggests this narrowing takes place near the opening of the pore on the inside of the cell. Other evidence suggests that it is between two vestibules. At any rate, CFTR appears selective for anions which are more easily dehydrated and follow the Lyotropic series, although it is not quite that simple. CFTR is considered a complex channel because it appears to have a variety of mechanisms for selectively passing chloride thru its pore, including distinct binding sites. The channel is also capable of discriminating between different ions based on their size.
In addition to a large vestibule on the intracellular side of the channel, there is evidence that a second vestibule exists near the extracellular side, since large MTS reagents are accessible when added to the extracellular side. The narrowest part of the pore could be the part between the vestibules. Channel-lining water-accessible residues have been found in TMs 1, 3, and 6 using the "substituted cysteine accessibility method". Also by this method, it was found that three amino acid residues, Glycine91, Lysine95, and Glutamine98 were lining the pore; all are from TM1. This sequence suggests a definite alpha-helical secondary structure. Some studies suggest that only TM domains 5, 6, 11, and 12 may be the helices needed to form the pore. McCarty et al in June, 2000 used a computer program to predict the three dimensional structure of the pore using all available mutagenesis data and found that these four TM domains could form the pore and that these helices would be arranged as tilted alpha-helices (like poles of a teepee). They hypothesize that the region where the poles of the teepee come together form the selectivity pore. And the large area underneath (corresponding to the living quarters of the teepee) is the large intracellular vestibule. And the area above where the poles come together (i.e. the chimney of the teepee) forms the smaller outer vestibule. This proposed structure reminds one of the structure of the potassium ion channel KscA which was solved back in 1998, but without any beta-sheet structures in the pore.
So it looks like once the chloride ion moves into the pore of CFTR, it sheds most or all of it's bound water molecules. It then binds to a positively charged amino acid in the large vestibule nearer the inside of the cell. This first stop may be part of the selection process, because larger organic anions like gluconate bind here, but do not usually go any further. The chloride ion will encounter at least one and possibly two regions inside the pore that are so narrow that nothing except a bare chloride ion may go thru. The ion will probably also bind at least two more positively charged amino acids before it makes its way out of the pore. All ion channels sequenced to date have at least one arginine amino acid in their predicted pore structures. And arginines are known from solution NMR studies to bind specifically to halide ions like chloride. Lysine95 (TM1) and Lysine335 (TM6) were the first two mutations deliberately engineered into CFTR to show any importance in anion selectivity. Changing them to negatively charged residues results in a preference for I- over Cl- ions. Lysine 347 in TM6 is also important for anion selectivity.
The amino acid proline99 in TM1 near the extracellular side of CFTR could also be part of the pore. When mutated to a leucine, there was no longer any selection for Cl- over I-. Sheppard and Welsh proposed that Proline 99 (on outside side of TM1) is involved in forming a kink in TM1 inside the channel, but may not itself line the pore. The pore of CFTR seems more selective for chloride (over iodide) ions when these ions are on the outside of the cell, but if the ions are on the inside of the cell, CFTR seems to prefer to let pass iodide over chloride. It has been suggested that a "pore loop" formed by amino acids from TM 6 near the intracellular side is responsible for an apparent narrowing as well.
There is a problem with this drawbridge analogy being used to describe opening and closing the pore which needs to be addressed. The vast majority of drugs found so far which block ion channels bind in a "voltage-dependent manner", meaning their binding to the pore depends on the overall conformation of the ion channel and probably not just a small part of the pore. The point being that there may be more involved in gating than a simple model presented here where only a small section of the pore is blocked off. It may turn out that major conformational changes take place in the pore which in turn affect gating.
Like all human ion channels discovered to date, CFTR needs to be strictly regulated by the body. A dramatic example of a breakdown in this type of communication has come to light by studies of the bacterium that causes cholera, Vibro cholerae. This microbe is still a serious threat in the world in large part because it is able to produce a toxin which keeps CFTR in the "on" position indefinitely. It is almost as if the bacteria is able to (literally) open the flood gates in the intestines using this toxin. The toxin modifies a Gs type of G-protein so that it is always able to indirectly activate CFTR by increasing cAMP. As a result of this attack by the toxin on the intestinal epithelia cell lining, overactive CFTR in those cells causes life-threatening diarrhea. Cholera victims have been known to die within several hours of first developing symptoms if not properly treated with rehydration therapy, due to massive and severe dehydration resulting from this uncontrolled diarrhea due to over-stimulated CFTR. The cholera bacterium has come upon this particular strategy and has used it to its advantage in order to flush itself out of the body of the host and into nearby water supplies, where it can infect more victims. CFTR is the rate-limiting step in water secretion by the intestines and therefore it is important for the body that it be closely regulated. In the lungs, CFTR regulation is also important, but for different reasons.
Now for the last part of this discussion: the shutting down of CFTR channel activity. At least two processes occur to cause the deactivation of CFTR: The first event is de-phosphorylation of the R-domain. The phosphate groups are removed by enzymes called phosphatases, which are co-localized to the cell membrane along with CFTR. Second, the pore must close by interactions with the intracellular domains. This is probably a result of ATP hydrolysis by NBD2. One of the best pieces of evidence for NBD2 involvement in channel closing comes from mutation studies. Changes in a single amino acid, a lysine to a methionine at position 1250 (i.e. K1250M) in NBD2 alters gating such that channels stay open longer than normal. It is because this mutant has a much harder time hydrolyzing ATP at NBD2.
The amino acid sequence LSGGQ in the NBD domains is known as the ABC "signature sequence", and serves to distinguish ABC family members like CFTR from other ATP hydrolyzing proteins. This sequence is found between the Walker A and B motifs in both NBD1 and NBD2 and may be important in coupling ATP hydrolysis with gating. The second glycine is the site of a natural known mutation in Cystic Fibrosis. LSGGQ is located in a section of NBD2 that is not part of the catalytic site and may therefore be free to interact with the transmembrane helices constituting the pore of CFTR. NBD2 may also be interacting directly with NBD1 to close the channel as well. Cross-talk between the two domains would not be surprising if it turns out to occur. Small molecules similar to ATP (but are different in being non-hydrolyzable by NBD2), probably bind to NBD2 and inhibit channel closing by inhibiting hydrolysis. This implies hydrolysis is necessary for channel closing. The NBD2 domain may normally act as a kind of "timer" switch, telling the channel when to shut off.
When the NBD2 domain "decides" to hydrolyze its bound ATP, this hydrolysis may act to change the conformation of NBD2. This new conformation could conceivably open up a new binding site for the R-domain, which may in turn change the conformation of the R-domain and make it a better substrate for the phosphatases. These phosphatases appear to always be on the lookout for proteins it can remove phosphate groups from. In other words, they are "constitutively active". When the R-domain in wild-type CFTR is unphosphorylated, it is somehow able to inhibit the NBD1 domain, perhaps by directly keeping it from either binding or hydrolyzing ATP. It may also "get in the way" of the NBD1 as the NBD tries to open the channel pore again like it normally would try to do during channel activation. Binding and cleavage of ATP most likely must occur at the NBD1 for the channel to open all the way to chloride. If the de-activated R-domain can inhibit this, it can inhibit channel opening again by NBD1. It is believed by many investigators in this field that the de-phorphroylated R-domain probably acts more to prevent ATP hydrolysis rather than ATP binding of NBD1. It is also likely that kinases other than the PKA (PKA if you remember was the kinase which first activated the R-domain) are able to phosphorylate specific inhibitory regions of the R-domain other than where PKA added phosphates to, such as a tyrosine residue. And this phosphorylation of a tyrosine also helps keep the R-domain from interacting in a positive way with the NBD1 domain.
CFTR is now closed and ready to become activated again the next time it is needed by another round of cyclic AMP stimulated PKA phosphorylation of the R-domain.
Summary Diagram: + = interactions which help open the pore - = interactions which help close pore
Some More Questions
Is R+ in ECL1 involved in gating by sensing extracellular pH? Does ECL1 affect gating (which takes place intracellularly) by an allosteric mechanism? Pseudomonas and Salmonella pathogenic bacteria bind ECL1. This loop may also influence which anions go thru the channel pore.
Does ICL1 interact directly with R-domain? How about the NBDs? There's little doubt it (and ICL2) are somehow involved in the gating mechanism because there's a decreased open probability of channels lacking them. They may affect conductance as well, and ICL1 is predicted to have 2 highly conserved alpha helices and is involved in gating the channel. ICL1 and ICL2 helps guide (target) CFTR to correct place in membrane during processing. ICL1 and ICL2 appear to help open the pore somehow. All of this fits in with the fact that there are so many CF-causing mutations in the intracellular loops, similarly to the NBDs.
ICL3 mutations affect conductance properties like rectification. Why does it appear to have cyclic nucleotide binding sites? Are they also involved in keeping the pore open? Both ICL3 and ICL4 appear to do this. Gating of the pore is definitely affected by mutations in ICL4. It's been speculated that the 4th intracellular loop is involved in coupling the activity of the NBDs to the pore.
Deletion of the TRL motif on the C-terminal end of CFTR has resulted in the mislocalization of CFTR in human airway cells epithelial cells. PDZ-containing proteins help couple CFTR to other regulatory proteins in the cell such as phosphatases, cytoskeletal proteins, or kinases, via the C-terminal of CFTR. Multivalency allows PDZ-domain containing proteins to provide scaffold-like places for protein-protein interactions, usually seen with signal transduction machinery, or the clustering of protein (ion channels, for example) at the cell membrane. It has been suggested that the PDZ proteins are the long-sought mechanism CFTR uses to regulate other ion channels.
All chloride channels including CFTR sequenced to date have at least one arginine in predicted pore, presumably to bind a chloride ion. Arginine amino acids on the outside of BSA protein are known to bind chloride ions specifically in solution (NMR studies). TM helices 1 thru 6 as well as 12 have the best chance of turning out to be involved in forming the pore. R352 may be involved in forming a pore loop which has the responsibility of helping with anion selectivity, while the rest of the M6 is in the alpha-helical form. Arg347 mutations alter both conductance and changes CFTR to a single anion pore (it is usually a multi-ion pore)
CFTR appears to have a relatively large vestibule within its pore near the intracellular side of the membrane. It is here that larger anions compete for a common binding site, probably an arginine amino acid residue.
Intracellular loops of CFTR are probably involved in channel gating as well as formation of part of the pore. They are longer than the extracellular loops which would make it all the more surprising if they were not involved at all. When measuring the conductance of CFTR without 30 amino acids in intracellular loop 1 in intracellular vesicles, CFTR shows non-wild type properties, including more subconductance state behavior (6 and 3pS) along with a reduced open probability to the wild type conductance of 8 pS. Generally, mutations introduced into the intracellular loops 1 and 2 tend to affect the pore such that there was an increased closed time, while mutations in intracellular loops 3 and 4 seem to be involved in decreasing the time the channel is open. Perhaps 1 and 2 help open the pore and 3 and 4 help keep it open. It's speculated that these loops help couple the NBDs to channel gating.
The boundaries of the transmembrane helices are clearly marked by amino acids which have low propensity for alpha helix or beta sheet formation. The boundary amino acids tend to have a high propensity for beta turn or irregular secondary structures. CFTR missing the first 118 amino acids functioned similarly to wild-type, but had a smaller conductance and open probability. Mutants without helix one can therefore form chloride channels.
Recent Evidence: More Pieces to the Puzzle?
Wang W, et al at the Alfred I. duPont Hospital for Children in Wilmington,
Delaware attempted to identify potential sites of domain-domain interaction
within CFTR by expressing "..purified, and refolded histidine (His)- and
glutathione-S-transferase (GST)-tagged cytoplasmic domains of CFTR."
They used tryptophan fluorescence quenching to detect an interaction between
NBD1-R and NBD2. They write that "binding among all pairwise
combinations of R-domain, NBD1, and NBD2 was demonstrated with an overlay
assay. To identify specific sites of interaction between
domains of CFTR, an overlay assay was used to probe an overlapping peptide
library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and
GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to
other intracellular domains onto crystal structures for HisP, MalK, and Rad50,
probable sites of interaction between NBD1 and NBD2 were identified.
Our data support a model where NBDs form dimers with the ATP-binding sites at
the domain-domain interface." Am J Physiol Cell Physiol 2002
May;282(5):C1170-80
In January, 2002, Cobb, BR. et al. reported finding that "PLA(2) and protein kinase A both contribute to A(2) receptor activation of CFTR, and components of this signaling pathway can augment wild-type and mutant CFTR activity" Am J Physiol Lung Cell Mol Physiol 2002 Jan;282(1):L12-25
In the recently published structure of an ABC superfamily member, MsbA, it can be seen that there is a central chamber within the membrane bilayer. It should be remembered that MsbA is a lipid pump from a bacteria rather than an ion channel (e.g. CFTR), however it is interesting to observe that its transmembrane helices, which form the chamber, are all tilted like a "teepee", with a tilt of between 30 and 40 degrees per helix. There are also two openings in the chamber which appear to allow lipid from the inner leaflet of the membrane to diffuse into the chamber, persumably to be pumped out to the other side of the bilayer once the NBD domains hydrolyze ATP. Chang and Roth, Science 293, 9/7/01 pgs 1793-1800.
In Septermer, 2001, Fu and Kirk (of the University of Alabama-Birmingham) determined that the inhibitory effect of the N-terminal tail of CFTR is due to the loss of negative charge and that the amino-terminal tail is also able to modulate other aspects of channel gating. They introduced cysteines at two positions (E54C/D58C) and then tested a series of methanethiosulfonate (MTS) reagents for their effects on the gating properties of these cysteine mutants in intact Xenopus oocytes and excised membrane patches. They reported that "Covalent modification of these sites with either neutral (MMTS) or charged (MTSET)) reagents markedly inhibited channel open probability primarily by reducing the rate of channel opening. The MTS reagents had negligible effects on the gating of the wild type channel or a corresponding double alanine mutant (E54A/D58A) under the same conditions. The inhibition of the opening rate of the E54C/D58C mutant channel by MMTS could be reversed by the reducing agent dithiothreitol or by elevating the bath ATP concentration above that required to activate maximally the wild type channel. Interestingly, the three MTS reagents had qualitatively different effects on the duration of channel openings (i.e. channel closing rate), namely the duration of openings was negligibly changed by the neutral MMTS, decreased by the positively charged MTSET, and increased by the negatively charged MTSCE." They concluded by saying their results indicated that "...the CFTR amino tail modulates both the rates of channel opening and channel closing and that the negative charges at residues 54 and 58 are important for controlling the duration of channel openings." J Biol Chem 2001 Sep 21;276(38):35660-8
In September, 2001, Ramjeesingh et al. (Hospitol for Sick Childre, Ontario
CA) determined that the normal function of CFTR is as a monomer, even though it
may be found as a homodimer in cell membranes such as CHO and Sf9 cells.
They noted that previous studies failed to identify the quaternary structure of
CFTR required to mediate chloride conduction and catalysis (ATP
hydrolysis). In their article, they stated that "...CFTR
molecules may self-associate in CHO and Sf9 membranes, as complexes close to the
predicted size of CFTR dimers can be captured by chemical cross-linking reagents
and detected using nondissociative PAGE. However, CFTR
function does not require a multimeric complex for function as we determined
that purified, reconstituted CFTR monomers are sufficient to mediate regulated
chloride conduction and ATPase activity." Biochemistry 2001 Sep
4;40(35):10700-6
In August, 2001 Falcon-Perez et al from the Instituto de Investigaciones
Biomedicas in Madrid, Spain reported that, in an effort to identify
intramolecular interactions in ABC Transporters necessary for energy transfer
mechanisms during transport, they made a series of mutant yeast cadmium factor
ABC proteins (ycf1) which would suppress ycf1 mutations that affect highly
conserved sites "presumably involved in ATP binding and/or
hydrolysis." They found thirteen intragenic second-site
suppressors ".... identified for the D777N mutation which affects the
invariant Asp residue in the Walker B motif of the first nucleotide binding
domain (NBD1)." They found that two of the suppressor
mutations (V543I and F565L) were located in the first transmembrane domain
(TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L,
and W1225C) are found within TMD2, one (S674L) in NBD1, and another one (R1415G)
is in NBD2, indicating either physical proximity or functional interactions
between NBD1 and the other three domains. They concluded
that "....the original D777N mutant protein exhibits a strong defect in the
apparent affinity for ATP and V(max) of transport. The phenotypic
characterization of the suppressor mutants shows that suppression does not
result from restoring these alterations but rather from a change in substrate
specificity." In the article, they discuss the possible
involvement of Asp777 in coupling ATPase activity to substrate binding and/or
transport across the membrane. Ycf1p is a vacuolar ATP binding
cassette (ABC) transporter required for heavy metal and drug detoxification and
shows strong sequence similarity to CFTR. J Bacteriol 2001
Aug;183(16):4761-70
In 2001, Nagel, Szellas, Riordanm Friedrich, and Hartung reported, using transport assays with the radioisotope sodium-22, that they have found evidence that CFTR does not directly activate the sodium channel ENaC, as has been assumed, but instead controls its activity indirectly by changing the cell membrane potential. CFTR could do this simply by transporting chloride. ENaC activity was dependent upon extracellular concentrations of chloride, and CFTR's effect on ENaC activation could be mimicked by other chloride channels. They concluded that "... these findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR. PMID: 11266369 2001
Chen, Chang, Aleksandrov and Riorden presented evidence in February, 2001 that CFTR probably functions as a monomer. They used several strategies, including cross-linking, co-immunoprecipitation, and single channel measurements to show that CFTR probably isn't normally a dimer when functioning properly. Biophysical Society Meeting, 2/2001
Choi, et al, (from The University of Texas Southwestern Medical Center, Dallas) Explored the nature of bicarbonate (HCO3-) secretion in CF verses normal tissues. They found that "...CFTR regulates other transporters, including Cl(-)-coupled HCO3- transport. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues, indicating the importance of this activity. HCO3- and pH affect mucin viscosity and bacterial binding." They examined chloride-coupled HCO3- transport by CFTR mutants that retain substantial or normal Cl- channel activity, and showed that "mutants reported to be associated with CF with pancreatic insufficiency do not support HCO3- transport, and those associated with pancreatic sufficiency show reduced HCO3- transport. Our findings demonstrate the importance of HCO3- transport in the function of secretory epithelia and in CF." Nature 2001 Mar 1;410(6824):94-7.
"It is generally believed that cAMP-dependent phosphorylation is the
principle mechanism for activating cystic fibrosis transmembrane conductance
regulator (CFTR) Cl(-) channels. However, we showed that activating G proteins
in the sweat duct stimulated CFTR Cl(-) conductance (G(Cl)) in the presence of
ATP alone without cAMP. The objective of this study was to test whether the G
protein stimulation of CFTR G(Cl) is independent of protein kinase A. We
activated G proteins and monitored CFTR G(Cl) in basolaterally permeabilized
sweat duct. Activating G proteins with guanosine 5'-O-(3-thiotriphosphate)
(10-100 microM) stimulated CFTR G(Cl) in the presence of 5 mM ATP alone without
cAMP. G protein activation of CFTR G(Cl) required Mg(2+) and ATP hydrolysis
(5'-adenylylimidodiphosphate could not substitute for ATP). G protein activation
of CFTR G(Cl) was 1) sensitive to inhibition by the kinase inhibitor
staurosporine (1 microM), indicating that the activation process requires
phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors
2',5'-dideoxyadenosine (1 mM) and SQ-22536 (100 microM); and 3) independent of
Ca(2+), suggesting that Ca(2+)-dependent protein kinase C and Ca(2+)/calmodulin-dependent
kinase(s) are not involved in the activation process. Activating AC with 10(-6)
M forskolin plus 10(-6) M IBMX (in the presence of 5 mM ATP) did not activate
CFTR, indicating that cAMP cannot accumulate sufficiently to activate CFTR in
permeabilized cells. We concluded that heterotrimeric G proteins activate CFTR
G(Cl) endogenously via a cAMP-independent pathway in this native absorptive
epithelium." Reddy, and Quinton. University of
California, San Diego : Am J Physiol Cell Physiol 2001 Mar;280(3):C604-13
"The cystic fibrosis transmembrane conductance regulator (CFTR) has been
shown previously to be regulated by inhibitory G proteins. In the present study,
we demonstrate inhibition of CFTR by alphaG(i2) and alphaG(i1), but not
alphaG(0), in Xenopus oocytes. We further examined whether regulators of G
protein signaling (RGS) proteins interfere with alphaG(i)-dependent inhibition
of CFTR. Activation of CFTR by IBMX and forskolin was attenuated in the presence
of alphaG(i2), indicating inhibition of CFTR by alphaG(i2) in Xenopus oocytes.
Coexpression of the proteins RGS3 and RGS7 together with CFTR and alphaG(i2)
partially recovered activation by IBMX/forskolin. 14-3-3, a protein that is
known to interfere with RGS proteins, counteracted the effects of RGS3. These
data demonstrate the regulation of CFTR by alphaG(i) in Xenopus oocytes. Because
RGS proteins interfere with the G protein-dependent regulation of CFTR, this may
offer new potential pathways for pharmacological intervention in cystic
fibrosis. Copyright 2001 Academic Press." Schreiber, Kindle,
Benzing, Walz, and Kunzelmann (University of Queensland, St. Lucia,
Queensland, 4072, Australia) : Biochem Biophys Res Commun 2001 Mar
9;281(4):917-23
One of the paradigms used to describe how the R-domain might affect CFTR channel
opening and closing has been based upon channel gating of the potassium channel Shaker.
It has been believed that they open and close via the "ball and chain"
model, whereby an intracellular ball-like domain of the channel is able to move
up to the opening of the pore and close it by plugging
it. New evidence based upon site-directed mutagenesis
and crystallography by MacKinnon et al. has revealed that the N-terminal tail
portion of the "ball" domain alone is able to snake its way deeper
into the pore and block it from potassium travel. They show that
electrostatic and hydrophobic events drive the closing. The
amino terminus of the tail domain is positively charged at normal pH inside the
cell, and the central cavity inside the pore is organized to favor cation
binding. The rest of the tail peptide is hydrophobic and binds to
the rest of the interior of the cavity, which is also hydrophobic.
Because the R-domain of CFTR does not have a free N-terminal peptide tail as the
potassium channel does, this may mean a rethinking of how the R-domain of CFTR,
when unphosphorylated, is able to block chloride ion flow. Nature
411, 657(2001).
Some references used for this page....
