How are the CFTR and the MRP different? (Or what are their substrates?)
07/06/10 07:20
MRP proteins are 50% homologous to the CFTR protein. That means that, when you look at the amino acids that go to make up the proteins, you see a 50% similarity in them. That structure of the protein dictates it’s function. It’s like it has “arms” inside of the cell, that only certain things fit into, to be pumped to the outside of the cell. Those things, that fit into the “arms” of the CFTR are what I’ve been talking about for a while now: substrates. When the structure of the amino acids in the protein are different, when the protein is put together differently, it’s going to change it’s substrates, because those “arms” only “fit” around compounds shaped to fit into the “arms.” Think of it as a square peg fitting into a round hole. If the shape of the protein is too much different, the substrates have to be very different as well. But, if the shapes of the proteins are similar, then the substrates will also be similar.
Someone asked me today, in an email, if BITC would make their child taste less salty. I had to tell him that I did not know. But that got me to thinking about substrates again, and about the CFTR and the MRP proteins, themselves.
This is the thing, with the “salty” question—See the article below (and there’s another one using azithromycin, with which I’m not going to fill up this blog post.) These compounds, that are substrates of the MRP proteins, cause also chloride efflux. I don’t know, at what point, they do this, because they have other substrates, which are going to compete with chloride ions, dependent on the structure of the protein. But the CFTR, itself, also transports chloride anions ALONG with other substrates, such as glutathione. But how much? I don’t know. Are the amounts of chloride anions transported by each protein comparable? I don’t know. So, will the kid taste salty, if he takes BITC? I don’t know.
Biochem Pharmacol. 2004 Jul 15;68(2):253-61.
Increased chloride efflux in colchicine-resistant airway epithelial cell lines.
Dragomir A, Roomans GM.
Department of Medical Cell Biology, University of Uppsala, Box 571, 751 23 Uppsala, Sweden. anca.dragomir@medcellbiol.uu.se
Abstract
Colchicine has been proposed as a treatment to alleviate chronic lung inflammation in cystic fibrosis patients and clinical trials are ongoing. Our aim was to investigate whether chronic exposure of cystic fibrosis cells to colchicine can affect their ability to transport chloride in response to cAMP. Colchicine-resistant cells were selected by growing in medium containing nanomolar concentrations of the drug. While microtubuli were affected by acute exposure to colchicine, they appeared normal in colchicine-resistant cells. Colchicine-resistant clones had higher expression of multidrug resistance proteins compared to untreated cells. Cystic fibrosis transmembrane conductance regulator (CFTR) labelling by immunocytochemistry showed no significant changes. The intracellular chloride concentration and basal chloride efflux of the cystic fibrosis treated cells increased significantly compared with untreated cells, while for the cAMP-stimulated Cl-efflux there was no significant change. The results suggest that colchicine promotes chloride efflux via alternative chloride channels. Since this is an accepted strategy for pharmacological treatment of cystic fibrosis, the results strengthen the notion that colchicine would be beneficial to these patients.
Let’s think about glutathione adducts….Remember? I told you we were going to cover them.
Now, glutathione (GSH) adducts are compounds combined, bound, to GSH. And it is this form, an adduct, that is transported by the MRP proteins. What compounds bind to GSH and are transported by the MRP? Colchicine, yes. Maybe azithromycin, too. Certainly chemotoxins, [Lancet. 1997 Sep 6;350(9079):711-2.] but also, probably, the isothiocyanates. And, the oxidized form of GSH, glutathione disulfide, is also transported. What do all of these things have in common?
THEY ARE NOT ANIONIC COMPOUNDS. Adducts of GSH are not anionic. Neither is GSSG. Now, look again, at the Colchicine article above. See this: “…while for the cAMP-stimulated Cl-efflux there was no significant change. The results suggest that colchicine promotes chloride efflux via alternative chloride channels.” Since we know that colchicine is transported by the MRP protein [Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8822-6.], and we know that the CFTR is responsible for cAMP-stimulated chloride efflux (transport out of the cell), then one of two things must be true:
1. MRP transports chloride ions along with other non-anionic compounds, and/or
2. MRP controls chloride transport via alternative chloride channels—(non CFTR, of course, but not directly transported by the MRP, either.)
I have no idea which it is, or whether it is both. How MUCH chloride it is responsible for moving out, I have no idea. I do know that chloride transport is important to neutrophil function, ie., the killing of bacteria (See the abstract below):
J Leukoc Biol. 2008 Jun;83(6):1345-53.
The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils.
Painter RG, Bonvillain RW, Valentine VG, Lombard GA, LaPlace SG, Nauseef WM, Wang G.
Gene Therapy Program, Department of Medicine and Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Abstract
Chloride anion is essential for myeloperoxidase (MPO) to produce hypochlorous acid (HOCl) in polymorphonuclear neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN-mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride-deficient medium killed PsA poorly. However, as the chloride level was raised, the killing efficiency increased in a dose-dependent manner. By using specific inhibitors to selectively block NADPH oxidase, MPO, and cystic fibrosis transmembrane conductance regulator (CFTR) functions, neutrophil-mediated killing of PsA could be attributed to three distinct mechanisms: CFTR-dependent and oxidant-dependent; chloride-dependent but not CFTR- and oxidant-dependent; and independent of any of the tested factors. Therefore, chloride anion is involved in oxidant- and nonoxidant-mediated bacterial killing. We previously reported that neutrophils from CF patients are defective in chlorination of ingested bacteria, suggesting that the chloride channel defect might impair the MPO-hydrogen peroxide-chloride microbicidal function. Here, we compared the competence of killing PsA by neutrophils from normal donors and CF patients. The data demonstrate that the killing rate by CF neutrophils was significantly lower than that by normal neutrophils. CF neutrophils in a chloride-deficient environment had only one-third of the bactericidal capacity of normal neutrophils in a physiological chloride environment. These results suggest that CFTR-dependent chloride anion transport contributes significantly to killing PsA by normal neutrophils and when defective as in CF, may compromise the ability to clear PsA.
… but this is not the entire arsenal of the airways. It is highly engaged in CF, because of increased neutrophilic migration, but that is not something that should be, if you can pack some “big guns” in, elsewhere in the system.
I’m sorry to give you so many “I don’t know’s” in this blog post. That’s just the way it is, sometimes. In studying this disease, it is of first importance, the questions that you can ask. The more the merrier, as they say. It is begging for trouble if you decide on a course of inquiry and can’t get off of it because you think that you already have the answer. It is the pure idiot who thinks he/she knows everything.
Someone asked me today, in an email, if BITC would make their child taste less salty. I had to tell him that I did not know. But that got me to thinking about substrates again, and about the CFTR and the MRP proteins, themselves.
This is the thing, with the “salty” question—See the article below (and there’s another one using azithromycin, with which I’m not going to fill up this blog post.) These compounds, that are substrates of the MRP proteins, cause also chloride efflux. I don’t know, at what point, they do this, because they have other substrates, which are going to compete with chloride ions, dependent on the structure of the protein. But the CFTR, itself, also transports chloride anions ALONG with other substrates, such as glutathione. But how much? I don’t know. Are the amounts of chloride anions transported by each protein comparable? I don’t know. So, will the kid taste salty, if he takes BITC? I don’t know.
Biochem Pharmacol. 2004 Jul 15;68(2):253-61.
Increased chloride efflux in colchicine-resistant airway epithelial cell lines.
Dragomir A, Roomans GM.
Department of Medical Cell Biology, University of Uppsala, Box 571, 751 23 Uppsala, Sweden. anca.dragomir@medcellbiol.uu.se
Abstract
Colchicine has been proposed as a treatment to alleviate chronic lung inflammation in cystic fibrosis patients and clinical trials are ongoing. Our aim was to investigate whether chronic exposure of cystic fibrosis cells to colchicine can affect their ability to transport chloride in response to cAMP. Colchicine-resistant cells were selected by growing in medium containing nanomolar concentrations of the drug. While microtubuli were affected by acute exposure to colchicine, they appeared normal in colchicine-resistant cells. Colchicine-resistant clones had higher expression of multidrug resistance proteins compared to untreated cells. Cystic fibrosis transmembrane conductance regulator (CFTR) labelling by immunocytochemistry showed no significant changes. The intracellular chloride concentration and basal chloride efflux of the cystic fibrosis treated cells increased significantly compared with untreated cells, while for the cAMP-stimulated Cl-efflux there was no significant change. The results suggest that colchicine promotes chloride efflux via alternative chloride channels. Since this is an accepted strategy for pharmacological treatment of cystic fibrosis, the results strengthen the notion that colchicine would be beneficial to these patients.
Let’s think about glutathione adducts….Remember? I told you we were going to cover them.
Now, glutathione (GSH) adducts are compounds combined, bound, to GSH. And it is this form, an adduct, that is transported by the MRP proteins. What compounds bind to GSH and are transported by the MRP? Colchicine, yes. Maybe azithromycin, too. Certainly chemotoxins, [Lancet. 1997 Sep 6;350(9079):711-2.] but also, probably, the isothiocyanates. And, the oxidized form of GSH, glutathione disulfide, is also transported. What do all of these things have in common?
THEY ARE NOT ANIONIC COMPOUNDS. Adducts of GSH are not anionic. Neither is GSSG. Now, look again, at the Colchicine article above. See this: “…while for the cAMP-stimulated Cl-efflux there was no significant change. The results suggest that colchicine promotes chloride efflux via alternative chloride channels.” Since we know that colchicine is transported by the MRP protein [Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8822-6.], and we know that the CFTR is responsible for cAMP-stimulated chloride efflux (transport out of the cell), then one of two things must be true:
1. MRP transports chloride ions along with other non-anionic compounds, and/or
2. MRP controls chloride transport via alternative chloride channels—(non CFTR, of course, but not directly transported by the MRP, either.)
I have no idea which it is, or whether it is both. How MUCH chloride it is responsible for moving out, I have no idea. I do know that chloride transport is important to neutrophil function, ie., the killing of bacteria (See the abstract below):
J Leukoc Biol. 2008 Jun;83(6):1345-53.
The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils.
Painter RG, Bonvillain RW, Valentine VG, Lombard GA, LaPlace SG, Nauseef WM, Wang G.
Gene Therapy Program, Department of Medicine and Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Abstract
Chloride anion is essential for myeloperoxidase (MPO) to produce hypochlorous acid (HOCl) in polymorphonuclear neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN-mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride-deficient medium killed PsA poorly. However, as the chloride level was raised, the killing efficiency increased in a dose-dependent manner. By using specific inhibitors to selectively block NADPH oxidase, MPO, and cystic fibrosis transmembrane conductance regulator (CFTR) functions, neutrophil-mediated killing of PsA could be attributed to three distinct mechanisms: CFTR-dependent and oxidant-dependent; chloride-dependent but not CFTR- and oxidant-dependent; and independent of any of the tested factors. Therefore, chloride anion is involved in oxidant- and nonoxidant-mediated bacterial killing. We previously reported that neutrophils from CF patients are defective in chlorination of ingested bacteria, suggesting that the chloride channel defect might impair the MPO-hydrogen peroxide-chloride microbicidal function. Here, we compared the competence of killing PsA by neutrophils from normal donors and CF patients. The data demonstrate that the killing rate by CF neutrophils was significantly lower than that by normal neutrophils. CF neutrophils in a chloride-deficient environment had only one-third of the bactericidal capacity of normal neutrophils in a physiological chloride environment. These results suggest that CFTR-dependent chloride anion transport contributes significantly to killing PsA by normal neutrophils and when defective as in CF, may compromise the ability to clear PsA.
… but this is not the entire arsenal of the airways. It is highly engaged in CF, because of increased neutrophilic migration, but that is not something that should be, if you can pack some “big guns” in, elsewhere in the system.
I’m sorry to give you so many “I don’t know’s” in this blog post. That’s just the way it is, sometimes. In studying this disease, it is of first importance, the questions that you can ask. The more the merrier, as they say. It is begging for trouble if you decide on a course of inquiry and can’t get off of it because you think that you already have the answer. It is the pure idiot who thinks he/she knows everything.