Substrates of multidrug resistance-associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel

1. The effects of physiological substrates of multidrug resistance-associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel currents were examined using patch clamp recording from CFTR-transfected mammalian cell lines. 2. Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and β-estradiol 17-(β-D-glucuronide) (E₂17βG) caused a voltage-dependent block of macroscopic CFTR Cl^- currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (K(D)s) of 96 ± 10 and 563 ± 103 μM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with K(D)s of 453 ± 44 and 3760 ± 710 μM (at 0 mV) respectively. 3. Reducing the extracellular Cl^- concentration from 154 to 20 mM decreased the K(D) for block intracellular TLCS to 54 ± 1 μM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl^- permeation through CFTR by binding within the channel pore. 4. Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel. 5. The apparent affinity of block by TLCs is comparable to that of other well-known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore. 6. These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane.