Susan Burbridge

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

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. Application of lonidamine to the intracellular face of excised membrane patches caused a voltage‐dependent block of CFTR currents, with an apparent Kd of 58 μM at −100 mV. Block by lonidamine was apparently independent of channel gating but weakly sensitive to the extracellular Cl− concentration. 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. 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. These results identify and characterize lonidamine as a novel CFTR open channel blocker and provide important information concerning its molecular mechanism of action.

Legionellaceae in the potable water of Nova Scotia hospitals and Halifax residences.

Water was cultured from 39 of 48 hospitals (7 Halifax hospitals and 32 non-Halifax hospitals) in the province of Nova Scotia and from 90 residences (74 private dwellings, 16 apartments) in Halifax to determine the frequency of legionella contamination. Six of seven Halifax hospitals had Legionellaceae isolated from their potable water compared with 3 of 32 non-Halifax hospitals (P < 0.0001). Overall, 19 of 59 (32%) of the water samples from Halifax hospitals were positive for legionellae compared with 5 of 480 (1%) samples from non-Halifax hospitals (P < 0.0000). Five of the six positive Halifax hospitals had Legionella pneumophila serogroup 1 and 1 had L. longbeachae serogroup 2 recovered from their potable water. Legionella contamination was associated with older, larger (> or = 50 beds) hospitals with total system recirculation. These hospitals also had water with a higher pH and calcium content but lower sodium, potassium, nitrate, iron and copper content. Fourteen of the 225 (6.2%) water samples from Halifax residences were positive for legionellae -8% (6/74) of the single family dwellings were positive, compared with 25% (4/16) apartments. The positivity rate of 15.7% for the 19 electric hot-water heaters in Halifax homes was not significantly different from the 32% positivity for Halifax hospitals. L. longbeachae accounted for 2 of the 14 isolates of legionellae from Halifax homes.

Colonisation of the respiratory tract with Legionella pneumophila for 63 days before the onset of pneumonia

We report the case of a 70-year-old man who was admitted to hospital A 66 days before developing Legionella pneumophila pneumonia 6 days after open heart surgery at hospital C. The strain of L. pneumophila recovered from the patients sputum was of the same subtype (monoclonal antibody type, enzyme type, plasmid profile, and restriction endonuclease pattern) as a strain of L. pneumophila in the potable water supplied to the room where he stayed in hospital A. We conclude that the patients respiratory tract became colonised by L. pneumophila while he was in hospital A and persisted for at least 63 days until he developed pneumonia requiring antibiotic treatment while in hospital C.