Short communication
Block of CFTR-dependent chloride currents by inhibitors of multidrug resistance-associated proteins

https://doi.org/10.1016/j.ejphar.2007.01.051Get rights and content

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that belongs to the same family as multidrug resistance-associated proteins whose main function is to expel xenobiotics and physiological organic anions from the cell interior. Despite the overall structural similarity with these membrane proteins, CFTR is not an active transporter but is instead a Cl channel. We have tested the ability of known inhibitors of multidrug resistance-associated proteins to affect CFTR Cl currents. We have found that sulfinpyrazone, probenecid, and benzbromarone are also inhibitors of CFTR activity, with a mechanism involving blockage of the channel pore.

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 Clcurrents. 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)

  • M. Dean et al.

    The human ATP-binding cassette (ABC) transporter superfamily

    Genome Res.

    (2001)
  • C.E. Grant et al.

    Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs

    Cancer Res.

    (1994)
  • Cited by (13)

    • Corrector-mediated rescue of misprocessed CFTR mutants can be reduced by the P-glycoprotein drug pump

      2012, Biochemical Pharmacology
      Citation Excerpt :

      CFTR and P-gp are predicted to be structurally similar and utilize similar biosynthetic pathways because both are members of the ATP-Binding cassette (ABC) family of proteins that contain two transmembrane (TM) domains (TMDs) (each containing 6 TM segments) and two NBDs [17]. Unfortunately, benzbromarone is unsuitable for treatment of CF because it inhibits CFTR activity by blocking the channel [18]. We predicted that direct binding of specific correctors to a site that does not inhibit channel activity would yield a more stable form of ΔF508 CFTR when compared to rescue with indirect methods such as expression at low temperature.

    • Correctors promote maturation of Cystic Fibrosis Transmembrane conductance Regulator (CFTR)-processing mutants by binding to the protein

      2007, Journal of Biological Chemistry
      Citation Excerpt :

      We first tested the system by using a compound that binds within the CFTR channel pore (at the interface between the two TMDs) and to see whether it could block cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Benzbromarone is a CFTR inhibitor that blocks channel activity by occupying the pore (18). Accordingly, HEK 293 cells expressing mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR were preincubated with various concentrations of benzbromarone and then treated with M11M cross-linker.

    • Chemotherapy-induced resistance by ATP-binding cassette transporter genes

      2007, Biochimica et Biophysica Acta - Reviews on Cancer
    View all citing articles on Scopus
    View full text