Mutation-induced Blocker Permeability and Multiion Block of the CFTR Chloride Channel Pore

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is blocked by a broad range of anions that bind tightly within the pore. Here we show that the divalent anion Pt(NO2)42− acts as an impermeant voltage-dependent blocker of the CFTR pore when added to the intracellular face of excised membrane patches. Block was of modest affinity (apparent Kd 556 μM), kinetically fast, and weakened by extracellular Cl− ions. A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO2)42− ions. Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Current in the presence of Pt(NO2)42− increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO2)42− ions to the extracellular solution. This “punchthrough” was prevented by extracellular Cl− ions, reminiscent of a “lock-in” effect. Relief of block in F337A by Pt(NO2)42− permeation was only observed for blocker concentrations above 300 μM; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Cl− current. We interpret this effect as reflecting concentration-dependent permeability of Pt(NO2)42− in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO2)42− to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)42− ions within the pore promotes their permeation to the extracellular solution

Molecular mechanism of anion permeation through CFTR channel pore.

The proposed research in my lab has been focusing on the structure, function and molecular pharmacology of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel; and the interaction of CFTR chloride channels with other ion channels, receptors in submucosal gland cells. Chronic lung infection and deterioration of lung function are the major causes of morbidity and death in cystic fibrosis (CF), the most common inherited lethal disease in Caucasians. Although the genetic defect in CF was discovered in 1989-mutations in the gene encoding the CFTR, the mechanism by which CFTR mutations cause lung disease remain uncertain. We arc very interested in studying a number of mechanisms proposed to link the CF genotype to clinical disease, particularly in defective airway submucosal gland secretion, loss of CFTR regulation of other transporting proteins such as other chloride channels, potassium channels, aquaporins in a serous epithelial cells. Determination of the mechanism linking genotype to disease is of crilical importance in developing therapies to treat CF

Multiple inhibitory effects of Au(CN)2− ions on cystic fibrosis transmembrane conductance regulator Cl− channel currents

Lyotropic pseudohalide anions are potentially useful as high affinity probes of Cl− channel pores. However, the interaction between these pseudohalides and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel have not been described in detail. Here we show that Au(CN)2− ions applied to the intracellular face of membrane patches from stably transfected baby hamster kidney cells inhibit CFTR channel currents by at least two mechanisms, which can be distinguished at the single channel level or by inhibiting channel closure using 2 mm pyrophosphate. Low concentrations (< 10 μm) of Au(CN)2− significantly reduced CFTR channel open probability. This effect was apparently voltage insensitive, independent of extracellular Cl− concentration, and lost following exposure to pyrophosphate. Higher concentrations of intracellular Au(CN)2− caused an apparent reduction in unitary current amplitude, presumably due to a kinetically fast blocking reaction. This effect, isolated following exposure to pyrophosphate, was strongly voltage dependent (apparent Kd 61.6 μm at −100 mV and 913 μm at +60 mV). Both the affinity and voltage dependence of block were highly sensitive to extracellular Cl− concentration. We propose that Au(CN)2− has at least two inhibitory effects on CFTR currents: a high affinity effect on channel gating due to action on a cytoplasmically accessible aspect of the channel and a lower affinity block within the open channel pore. These results offer important caveats for the use of lyotropic pseudohalide anions such as Au(CN)2− as specific high affinity probes of Cl− channel pores

Submucosal gland ion channels and cystic fibrosis.

Cystic fibrosis (CF), the most common fatal genetic disease, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) [20, 22]. Since the CFTR gene was identified in 1989 [23], a lot of research effort has been focused on either replacing the defective gene or rescuing the function of mutant CFTR in CF patients [20, 22]. Overall, no currently approved treatment cures the disease [22]. Most of the morbidity and mortality of CF results from lung disease which involves devastating loss of transepithelial anion secretion [20, 28, 29]. In lung, CFTR is strongly expressed in submucosal glands, it has been suggested that these cells represent the primary site of cystic fibrosis pathology [1, 2]. Anion secretion in submucosal gland is mediated by CFTR cr channels at the apical membrane [4], however, other chloride channels may also involves and the net rate of secretion is determined by the activity of basolateral K+ channels.ARC 05/0

Optimisation and modelling of the growth and astaxanthin formation of haematococcus pluvialis

published_or_final_versionBotanyDoctoralDoctor of Philosoph

Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is blocked by highly lyotropic permeant anions which bind tightly within the pore. Here we show that several different substitutions of a positively charged amino acid residue, arginine R334, in the putative outer mouth of the CFTR pore, greatly reduce the block caused by lyotropic Au(CN)2− ions applied to the intracellular side of the channel. Fixed positive charge at this site appears to play a role in Au(CN)2− binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. However, non-charge-dependent effects also appear to contribute to Au(CN)2− binding. Mutation of R334 also disrupts the apparent electrostatic interaction between intracellular Au(CN)2− ions and extracellular permeant anions, an interaction which normally acts to relieve channel block. All six mutations studied at R334 significantly weakened this interaction, suggesting that arginine possesses a unique ability to coordinate ion-ion interactions at this site in the pore. Our results suggest that lyotropic anions bind tightly to a site in the outer mouth of the CFTR pore that involves interaction with a fixed positive charge. Binding to this site is also involved in coordination of multiple permeant anions within the pore, suggesting that anion binding in the outer mouth of the pore is an important aspect in the normal anion permeation mechanism

Molecular determinants of Au(CN)2− binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl− channel pore

Lyotropic anions with low free energy of hydration show both high permeability and tight binding in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel pore. However, the molecular bases of anion selectivity and anion binding within the CFTR pore are not well defined and the relationship between binding and selectivity is unclear. We have studied the effects of point mutations throughout the sixth transmembrane (TM6) region of CFTR on channel block by, and permeability of, the highly lyotropic Au(CN)2− anion, using patch clamp recording from transiently transfected baby hamster kidney cells. Channel block by 100 μm Au(CN)2−, a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Relative Au(CN)2− permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. These results are used to define a model of the pore containing multiple anion binding sites but a more localised anion selectivity region. The central part of TM6 (F337-S341) appears to be the main determinant of both anion binding and anion selectivity. However, comparison of the effects of individual mutations on binding and selectivity suggest that these two aspects of the permeation mechanism are not strongly interdependent

Mechanism of lonidamine inhibition of the CFTR chloride channel

1. The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(−) channel is blocked by a broad range of organic anionic compounds. Here we investigate the effects of the indazole compound lonidamine on CFTR channels expressed in mammalian cell lines using patch clamp recording. 2. Application of lonidamine to the intracellular face of excised membrane patches caused a voltage-dependent block of CFTR currents, with an apparent K(d) of 58 μM at −100 mV. 3. Block by lonidamine was apparently independent of channel gating but weakly sensitive to the extracellular Cl(−) concentration. 4. Intracellular lonidamine led to the introduction of brief interruptions in the single channel current at hyperpolarized voltages, leading to a reduction in channel mean open time. Lonidamine also introduced a new component of macroscopic current variance. Spectral analysis of this variance suggested a blocker on rate of 1.79 μM(−1) s(−1) and an off-rate of 143 s(−1). 5. Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) significantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. 6. These results identify and characterize lonidamine as a novel CFTR open channel blocker and provide important information concerning its molecular mechanism of action