Short communicationBlock of CFTR-dependent chloride currents by inhibitors of multidrug resistance-associated proteins
Introduction
The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane protein that belongs to the family of ATP-binding cassette (ABC) transporters (Dean et al., 2001, Schmitt and Tampe, 2002). Such membrane proteins share a similar architecture based on various transmembrane helices (from 12 to 17) and two nucleotide binding domains (NBD1 and 2). Interaction and hydrolysis of ATP at two sites in the NBDs induces conformational changes that drive active transport of various types of molecules across the plasma membrane (Dean et al., 2001, Schinkel and Jonker, 2003). CFTR is part of the subfamily C of ABC (ABCC) transporters which includes the multidrug resistance-associated proteins (Kruh and Belinsky, 2003). These proteins work as active transporters of endogenous substrates, like ABCC1 for LTC4 (Leier et al., 1994, Jedlitschky et al., 1994), and of exogenous substances, called xenobiotics. Such compounds are transported in their native state or as conjugates with glutathione (Ishikawa, 1992), glucunorate, or sulfates (Jedlitschky et al., 1996). In general, ABCC drug transporters have a preference for anionic compounds in contrast to the multidrug resistance protein 1, ABCB1, which is more selective for neutral or slightly basic compounds (Schinkel and Jonker, 2003). The wide spectrum of substances translocated by multidrug resistance proteins is beneficial because it provides protection against potentially toxic exogenous molecules (Leslie et al., 2001, Hipfner et al., 1999). However, many ABCC transporters, as well as ABCB1, are also responsible for the multidrug resistance shown by different types of human tumours (Grant et al., 1994, Kruh et al., 2001, Sawicka et al., 2004).
Among the ABCC subfamily, CFTR is the only protein that does not generate an active transport. In fact, CFTR is a plasma membrane Cl− channel (Anderson et al., 1991) in which the conformational changes generated by NBD/ATP interactions are not used for active transport but rather for the opening and closing of the pore (Sheppard and Welsh, 1999). However, there are still some intriguing findings that suggest that multidrug resistance-associated proteins and CFTR have some similarities beyond the amino acid sequence homology. For example, it has been reported by some investigators that CFTR is also able to translocate glutathione as done by other ABCC proteins (although by passive diffusion and not by active transport) (Linsdell and Hanrahan, 1998). Furthermore, substrates of multidrug resistance-associated proteins inhibit CFTR Cl− currents by interacting with the CFTR pore from the cytosolic side (Linsdell and Hanrahan, 1999). This suggests a common mechanism of interaction at the level of the transmembrane portion of the proteins.
We have tested the ability of known ABCC inhibitors to affect CFTR Cl−currents. This is important to further explore the analogies between CFTR and ABCC drug transporters and, possibly, to develop novel CFTR blockers which could be useful for the treatment of secretory diarrhea (Verkman et al., 2006). Our data show that sulfinpyrazone, probenecid, and, particularly, benzbromarone are effective inhibitors of the CFTR channel through a probable block of the pore.
Section snippets
Cell culture
Fischer rat thyroid (FRT) cells stably expressing human CFTR were cultured on plastic in Coon's modified F12 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. T84 cells were cultured in DMEM/F12 plus 10% fetal bovine serum, l-glutamine and antibiotics (same concentrations as for FRT cells).
Transepithelial Cl− currents
For short-circuit current measurements, cells were plated on Snapwell permeable supports (Corning–Costar) at 500,000 cells/Snapwell. After
Results
Fig. 1A shows the chemical structure of multidrug resistance-associated protein inhibitors compared to known CFTR blockers. It is evident that the compounds belong to different chemical classes. However, they have in common a negative electrical charge at physiological pH. The sensitivity of the CFTR channel towards inhibitors was determined by measuring transepithelial Cl− currents in transfected FRT cells. Maximal stimulation with the adenylyl cyclase activator, forskolin (20 μM), evoked
Discussion
CFTR is unique within the large family of ABC transporters because it is the only protein with ion channel function. Despite the differences in function, ABCC drug transporters and CFTR share some structural features. First of all, they have a high level of amino acid conservation at some critical residues in the NBDs. Furthermore, they have a similarity in the overall organization of the transmembrane domains. Our aim was to verify whether inhibitors of ABCC transporters have the ability to
Acknowledgments
This work was supported by Telethon-Italy (GGP05103), CIPE-Regione Liguria (Biofarma 2) and the NIH (P30 DK072517). We also thank Dr. Kerry Rhoden for kindly reading and correcting the manuscript.
References (24)
- et al.
Structural, mechanistic and clinical aspects of MRP1
Biochim. Biophys. Acta
(1999) - et al.
Molecular pharmacology of the CFTR Cl− channel
Trends Pharmacol. Sci.
(1999) The ATP-dependent glutathione S-conjugate export pump
Trends Biochem. Sci.
(1992)- et al.
The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates
J. Biol. Chem.
(1994) - et al.
Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters
Toxicology
(2001) - et al.
Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview
Adv. Drug Deliv. Rev.
(2003) - et al.
Structure and mechanism of ABC transporters
Curr. Opin. Struct. Biol.
(2002) - et al.
Demonstration that CFTR is a chloride channel by alteration of its anion selectivity
Science
(1991) - et al.
T84 cells: anion selectivity demonstrates expression of Cl− conductance affected in cystic fibrosis
Am. J. Physiol.
(1992) - et al.
CFTR: mechanism of anion conduction
Physiol. Rev.
(1999)
The human ATP-binding cassette (ABC) transporter superfamily
Genome Res.
Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs
Cancer Res.
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