Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be “transplanted” to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl− conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby “rescuing” the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl− conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO2)42− in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl− channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl− transport through a combination of failure to increase Cl− conductance and increased susceptibility to channel block by cytosolic substances.
The purpose of the present study was to determine whether a physiologic plasma concentration of a-ketoglutarate (aKG) influences the kinetic interaction of ligands with organic anion transporter 1 (OAT1). The effect of extracellular aKG on the kinetics of para-aminohippurate (PAH) and cidofovir transport was examined along with its effect on the potency of 10 drugs in five different classes (uricosuric, nonsteroidal anti-inflammatories, loop diuretics, angiotensin II receptor antagonists, and b-lactam antibiotics) to inhibit OAT1 expressed in Chinese hamster ovary cells. Extracellular aKG competitively inhibited PAH and cidofovir transport with K i values (∼5 mM) approximating its unbound plasma concentration (determined by equilibrium dialysis). When PAH was the substrate, extracellular aKG (5 mM) significantly increased IC 50 values for some inhibitors (up to 4-fold), such as probenecid, but not for others (an inhibitor-dependent effect). For some inhibitors, a significant increase in IC 50 value was observed when cidofovir was the substrate, but not PAH (a substrate-dependent effect). A significant increase in IC 50 value was also observed for inhibition of PAH transport by probenecid in renal basolateral membrane vesicles (5.2-fold). The substrate-and inhibitor-dependent effect of extracellular aKG on ligand interactions with OAT1 highlights the complexity of the OAT1 ligand-binding surface. The effect of extracellular aKG on the potency of OAT1 inhibition should be considered when assessing drug-drug interaction potential at the transporter.
Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.
Li MS, Holstead RG, Wang W, Linsdell P. Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate. Am J Physiol Cell Physiol 300: C65-C74, 2011. First published October 6, 2010 doi:10.1152/ajpcell.00290.2010.-The CFTR contributes to Cl Ϫ and HCO 3 Ϫ transport across epithelial cell apical membranes. The extracellular face of CFTR is exposed to varying concentrations of Cl Ϫ and HCO 3 Ϫ in epithelial tissues, and there is evidence that CFTR is sensitive to changes in extracellular anion concentrations. Here we present functional evidence that extracellular Cl Ϫ and HCO 3 Ϫ regulate anion conduction in open CFTR channels. Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO 3 Ϫ than when it contains Cl Ϫ . This difference appears to reflect differences in the ability of extracellular HCO 3 Ϫ and Cl Ϫ to interact with and repel intracellular blocking anions from the pore. Strong block by endogenous cytosolic anions leading to reduced CFTR channel currents in intact cells occurs at physiologically relevant HCO 3 Ϫ concentrations and membrane potentials and can result in up to ϳ50% inhibition of current amplitude. We propose that channel block by cytosolic anions is a previously unrecognized, physiologically relevant mechanism of channel regulation that confers on CFTR channels sensitivity to different anions in the extracellular fluid. We further suggest that this anion sensitivity represents a feedback mechanism by which CFTR-dependent anion secretion could be regulated by the composition of the secretions themselves. Implications for the mechanism and regulation of CFTR-dependent secretion in epithelial tissues are discussed. anion secretion; cystic fibrosis transmembrane conductance regulator; open channel block CYSTIC FIBROSIS (CF), a disease of deficient epithelial cell anion transport, is caused by genetic mutations that result in loss of function of the CFTR anion channel (15). CFTR is expressed in many different epithelial tissues, and, as a result, CF is associated with lung, pancreatic, gastrointestinal, and reproductive disease (45,46 (10,19,27). Again, the relative importance of direct HCO 3 Ϫ transport by CFTR vs.indirect, CFTR-regulated HCO 3 Ϫ transport by SLC26 proteins to overall epithelial cell HCO 3 Ϫ transport and their relevance to CF disease are the subject of current debate (24,29,51,55).Since CFTR is expressed in the apical membrane of epithelial cells, its extracellular face is exposed to epithelial secretions in the luminal fluid; the composition of this fluid may change dramatically under different conditions. The most striking example is the pancreatic duct, which in humans can secrete a fluid containing up to 140 mM HCO 3,5,7,13,25), and there is evidence that some epithelia may "switch" between Cl Ϫ and HCO 3 Ϫ secretion, depending on the stimulus (8,...
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