Substrates
17/05/10 09:17
I have studied, though the years, both the MRP and the CFTR proteins. How are they alike? How are they different? The fact that I’m turning over in my mind right now is that the CFTR is a multi-anion pore. It can hold more than one anion (a negatively charged ion, remember?) at a time, and it also transports more than just one type of anion.
The current literature tells us that it transports:
1. chloride ions (Cl-)
2. thiocyanate (SCN-)
3. bicarbonate (HCO3-)
4. glutathione (GSH)
But. Let’s look at the literature on MRP—
First of all, it is known for its role in detoxification processes. What are these processes?
Well, there’s Phase I, and Phase II, and then there’s Phase III antiporter processes. It is the latter two that we are interested in, when it comes to the function of MRP proteins. The Phase II detoxification process is, first and foremost, a conjugation process. (Conjugation is where two compounds are joined together, to form another compound.) That compound is then transported out of the cell by the Phase III antiporter process—it’s pumped out, in other words.
So, what this means is that something is conjugated with, most likely, an anion, and then the MRP pumps it out of the cell. Let’s address the current literature, to see if the above listed anions are used in this process:
Chloride – There’s not much on this topic—MRP (or MDR, which is sometimes used interchangeably, with MRP, to refer to multi-drug resistant proteins) and chloride transport. There is this:
Am J Physiol. 1996 May;270(5 Pt 1):C1370-8.
P-glycoprotein-associated chloride currents revealed by specific block by an anti-P-glycoprotein antibody.
Han ES, Vanoye CG, Altenberg GA, Reuss L.
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641, USA.
Abstract
The relationships between P-glycoprotein (PGP) expression and plasma membrane ion currents activated by cell swelling were studied in several cell lines by use of the whole cell configuration of the patch-clamp technique. Swelling-activated Cl- currents (ICls) had similar characteristics independently of whether PGP was expressed. Addition of the anti-PGP monoclonal antibody C219 or its Fab fragment to the pipette solution prevented ICls in cells expressing functional PGP (assessed by immunoblots, immunofluorescence, and transport of rhodamine 123) but not in cells lacking PGP expression. A peptide analogue of the C219 epitope abolished the effect of C219. Other anti-PGP antibodies and mouse immunoglobulin G were ineffective. C219 did not alter swelling-activated cation currents. Inasmuch as ICls is present in cells that do not express PGP and C219 has no effect on ICls in these cells, we conclude that PGP is not required for the ICls phenotype. However, when expressed in the plasma membrane, PGP is involved, directly or indirectly, in ICls but not in swelling-activated K+ currents.
And this:
Phys Med Biol. 2008 Jan 21;53(2):N1-7. Epub 2007 Dec 28.
Dielectrophoretic analysis of changes in cytoplasmic ion levels due to ion channel blocker action reveals underlying differences between drug-sensitive and multidrug-resistant leukaemic cells.
Duncan L, Shelmerdine H, Hughes MP, Coley HM, Hübner Y, Labeed FH.
Centre for Biomedical Engineering, School of Engineering (H5), University of Surrey, Guildford GU27XH, UK.
Abstract
Dielectrophoresis (DEP)--the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K(+) and Ca(2+) levels were extremely similar between sensitive and resistant lines, levels of Cl(-) were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function.
Which tells us that the multi-drug transport proteins are involved in ion transport, and probably chloride ion transport, but that’s not really the question that we want answered. We want to know if chloride is conjugated to any other compound, and then transported out. Chloride transport mediated by multidrug resistance proteins was of interest, first, in cancer therapy, when it was noticed that cancer patients who were given chemotoxins had changes in their volume activated chloride channel activity, but this tells us nothing about chloride as a conjugate. Perhaps it is simply transported by the MRP proteins, while the protein is transporting conjugates. In fact, that would seem to be the case—
Am J Physiol Lung Cell Mol Physiol. 2001 Jul;281(1):L24-30.
Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia.
Gao L, Broughman JR, Iwamoto T, Tomich JM, Venglarik CJ, Forman HJ.
Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Abstract
Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value),
whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.
Now, this doesn’t mean that chloride is moved out along with a conjugate, specifically. And it certainly does not mean that chloride is conjugated with anything else, and is then transported by the MRP protein. But what it does mean is that where glutathione goes, so go chloride ions. They share a transport mechanism; and probably are both substrates of the same transport protein.
I will leave you here, for now, with those thoughts. The next time, I will cover glutathione and it’s conjugates.
The current literature tells us that it transports:
1. chloride ions (Cl-)
2. thiocyanate (SCN-)
3. bicarbonate (HCO3-)
4. glutathione (GSH)
But. Let’s look at the literature on MRP—
First of all, it is known for its role in detoxification processes. What are these processes?
Well, there’s Phase I, and Phase II, and then there’s Phase III antiporter processes. It is the latter two that we are interested in, when it comes to the function of MRP proteins. The Phase II detoxification process is, first and foremost, a conjugation process. (Conjugation is where two compounds are joined together, to form another compound.) That compound is then transported out of the cell by the Phase III antiporter process—it’s pumped out, in other words.
So, what this means is that something is conjugated with, most likely, an anion, and then the MRP pumps it out of the cell. Let’s address the current literature, to see if the above listed anions are used in this process:
Chloride – There’s not much on this topic—MRP (or MDR, which is sometimes used interchangeably, with MRP, to refer to multi-drug resistant proteins) and chloride transport. There is this:
Am J Physiol. 1996 May;270(5 Pt 1):C1370-8.
P-glycoprotein-associated chloride currents revealed by specific block by an anti-P-glycoprotein antibody.
Han ES, Vanoye CG, Altenberg GA, Reuss L.
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641, USA.
Abstract
The relationships between P-glycoprotein (PGP) expression and plasma membrane ion currents activated by cell swelling were studied in several cell lines by use of the whole cell configuration of the patch-clamp technique. Swelling-activated Cl- currents (ICls) had similar characteristics independently of whether PGP was expressed. Addition of the anti-PGP monoclonal antibody C219 or its Fab fragment to the pipette solution prevented ICls in cells expressing functional PGP (assessed by immunoblots, immunofluorescence, and transport of rhodamine 123) but not in cells lacking PGP expression. A peptide analogue of the C219 epitope abolished the effect of C219. Other anti-PGP antibodies and mouse immunoglobulin G were ineffective. C219 did not alter swelling-activated cation currents. Inasmuch as ICls is present in cells that do not express PGP and C219 has no effect on ICls in these cells, we conclude that PGP is not required for the ICls phenotype. However, when expressed in the plasma membrane, PGP is involved, directly or indirectly, in ICls but not in swelling-activated K+ currents.
And this:
Phys Med Biol. 2008 Jan 21;53(2):N1-7. Epub 2007 Dec 28.
Dielectrophoretic analysis of changes in cytoplasmic ion levels due to ion channel blocker action reveals underlying differences between drug-sensitive and multidrug-resistant leukaemic cells.
Duncan L, Shelmerdine H, Hughes MP, Coley HM, Hübner Y, Labeed FH.
Centre for Biomedical Engineering, School of Engineering (H5), University of Surrey, Guildford GU27XH, UK.
Abstract
Dielectrophoresis (DEP)--the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K(+) and Ca(2+) levels were extremely similar between sensitive and resistant lines, levels of Cl(-) were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function.
Which tells us that the multi-drug transport proteins are involved in ion transport, and probably chloride ion transport, but that’s not really the question that we want answered. We want to know if chloride is conjugated to any other compound, and then transported out. Chloride transport mediated by multidrug resistance proteins was of interest, first, in cancer therapy, when it was noticed that cancer patients who were given chemotoxins had changes in their volume activated chloride channel activity, but this tells us nothing about chloride as a conjugate. Perhaps it is simply transported by the MRP proteins, while the protein is transporting conjugates. In fact, that would seem to be the case—
Am J Physiol Lung Cell Mol Physiol. 2001 Jul;281(1):L24-30.
Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia.
Gao L, Broughman JR, Iwamoto T, Tomich JM, Venglarik CJ, Forman HJ.
Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Abstract
Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value),
whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.
Now, this doesn’t mean that chloride is moved out along with a conjugate, specifically. And it certainly does not mean that chloride is conjugated with anything else, and is then transported by the MRP protein. But what it does mean is that where glutathione goes, so go chloride ions. They share a transport mechanism; and probably are both substrates of the same transport protein.
I will leave you here, for now, with those thoughts. The next time, I will cover glutathione and it’s conjugates.