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

1 The effects of physiological substrates of multidrug resistance-associate d proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (C FTR) Cl- channel currents were examined using patch clamp recording from CF TR-transfected mammalian cell lines. 2 Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and beta-estradio l 17-(beta-D-glucuronide) (E(2)17 beta G) 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 mu M (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel block ers under these conditions, with K(D)s of 453 +/- 44 and 3760 +/- 710 mu M (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 mu M, and also significant ly 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 th e gating of the channel. 5 The apparent affinity of block by TLCs is comparable to that of other wel l-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 sha re a structurally similar anion binding site at the cytoplasmic face of the membrane.