Gating of the CFTR Cl− channel by ATP-driven nucleotide-binding domain dimerisation

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl⁻ channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating.     

Glucagon activates Ca2+ and Cl− channels in rat hepatocytes

Glucagon is one of the major hormonal regulators of glucose metabolism, counteracting the hepatic effects of insulin when the concentration of glucose in the bloodstream falls below a certain level. Glucagon also regulates bile flow, hepatocellular volume and membrane potential of hepatocytes. It is clear that changes in cell volume and membrane potential cannot occur without significant ion fluxes across the plasma membrane. The effects of glucagon on membrane currents in hepatocytes, however, are not well understood. Here we show, by patch-clamping of rat hepatocytes, that glucagon activates two types of currents: a small inwardly rectifying Ca²⁺ current with characteristics similar to those of the store-operated Ca²⁺ current and a larger outwardly rectifying Cl⁻ current similar to that activated by cell swelling. We show that the mechanism of glucagon action on membrane conductance involves phospholipase C and adenylyl cyclase. Contribution of the adenylyl cyclase-dependent pathway to activation of the currents depended on Epac (exchange protein directly activated by cAMP), but not on protein kinase A. The activation of Ca²⁺ and Cl⁻ channels is likely to play a key role in the mechanisms by which glucagon regulates hepatocyte metabolism and volume.     

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 μ m 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.     

Activation of ferret erythrocyte Na+–K+–2Cl− cotransport by deoxygenation

Deoxygenation of ferret erythrocytes stimulates Na⁺–K⁺–2Cl⁻ cotransport by 111% ( s.d. , 46) compared to controls in air. Half-maximal activation occurs at a P _O2 of 24 mmHg ( s.d. , 2) indicating that physiological changes in oxygen tension can influence cotransport function. Approximately 25–35% of this stimulation can be attributed to the rise of intracellular free magnesium concentration that occurs on deoxygenation (from 0.82 ( s.d. , 0.07) to 1.40 m m ( s.d. , 0.17)). Most of the stimulation is probably caused by activation of a kinase which can be prevented or reversed by treating cells with the kinase inhibitors PP1 or staurosporine, or by reducing cell magnesium content to submicromolar levels. Stimulation by deoxygenation is comparable with that caused by calyculin A or sodium arsenite, compounds that cause a 2- to 3-fold increase in threonine phosphorylation of the cotransporter which can be detected with phospho-specific antibodies. However, the same approach failed to detect significant changes in threonine phosphorylation following deoxygenation. The results suggest that deoxygenation causes activation of a kinase that either phosphorylates the transporter, but probably not on threonine, or phosphorylates another protein that in turn influences cotransporter behaviour. They also indicate that more than one kinase and phosphatase are involved in cotransporter phosphorylation.     

Cyclic AMP-dependent Cl− secretion induced by thromboxane A2 in isolated human colon

Increased release of thromboxane A₂ (TXA₂) has been shown to be involved in inflammatory bowel diseases. In the present study, we have investigated the effect of a stable TXA₂ analogue (STA₂) on the electrical parameters in isolated human colonic mucosa. In the human mucosa set between Ussing chambers, STA₂ stimulated Cl⁻ secretion in a concentration-dependent manner with an EC₅₀ of 0.06 μ m . The STA₂-induced Cl⁻ secretion was significantly inhibited by ONO-3708 (10 μ m ), a specific TXA₂ receptor antagonist. The effect of STA₂ (0.3 μ m ) was independent of the colonic segment from which the tissue was obtained, from caecum to rectum. Chromanol 293B, an inhibitor of the cAMP-dependent KvLQT1 channel, attenuated the STA₂-induced Cl⁻ secretion in the human colonic mucosa (IC₅₀ value 1.18 μ m ). We found that KvLQT1 mRNA and protein were expressed in all the tested segments of the human colon. The STA₂-induced Cl⁻ secretion was significantly inhibited by 8-bromo-2'-monobutyryladenosine-3',5'-cyclic monophosphorothioate (50 μ m ), a membrane-permeant cAMP antagonist. STA₂ (0.3 μ m ) significantly increased the intracellular cAMP levels and the short-circuit current via TXA₂ receptor in a human colonic cell line. These results suggest that the TXA₂-induced Cl⁻ secretion in the colon is mediated via the cAMP pathway in addition to the Ca²⁺–calmodulin pathway which was previously reported.