Imaging CFTR in its native environment

Schillers, Hermann

doi:10.1007/s00424-007-0399-8

Cited by 28 publications

(26 citation statements)

References 114 publications

(82 reference statements)

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“…Electron microscope studies of purified, antibodydecorated CFTR performed at 2-nm resolution revealed CFTR as a dimer of ellipsoidal form (12 9 10.6 9 16.2 nm) with orifices beneath the putative TMD [28]. Atomic force microscopy (AFM) of immunogold-labeled CFTR on isolated plasma membranes also revealed the CFTR channel as a tail-to-tail dimer with a central pore of unrealistically large (*10 nm) radius [32]. In contrast, single molecule fluorescence imaging in live cells [33] and electron microscopy of two-dimensional CFTR crystals [26] provided evidence for monomeric CFTR.…”

Section: Introductionmentioning

confidence: 95%

ATP hydrolysis-dependent asymmetry of the conformation of CFTR channel pore

2011

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Despite substantial efforts, the entire cystic fibrosis transmembrane conductance regulator (CFTR) protein proved to be difficult for structural analysis at high resolution, and little is still known about the actual dimensions of the anion-transporting pathway of CFTR channel. In the present study, we therefore gauged geometrical features of the CFTR Cl(-) channel pore by a nonelectrolyte exclusion technique. Polyethylene glycols with a hydrodynamic radius (R (h)) smaller than 0.95 nm (PEG 300-1,000) added from the intracellular side greatly suppressed the inward unitary anionic conductance, whereas only molecules with R (h) ≤ 0.62 nm (PEG 200-400) applied extracellularly were able to affect the outward unitary anionic currents. Larger molecules with R (h) = 1.16-1.84 nm (PEG 1,540-3,400) added from either side were completely excluded from the pore and had no significant effect on the single-channel conductance. The cut-off radius of the inner entrance of CFTR channel pore was assessed to be 1.19 ± 0.02 nm. The outer entrance was narrower with its cut-off radius of 0.70 ± 0.16 nm and was dilated to 0.93 ± 0.23 nm when a non-hydrolyzable ATP analog, 5'-adenylylimidodiphosphate (AMP-PNP), was added to the intracellular solution. Thus, it is concluded that the structure of CFTR channel pore is highly asymmetric with a narrower extracellular entrance and that a dilating conformational change of the extracellular entrance is associated with the channel transition to a non-hydrolytic, locked-open state.

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Section: Introductionmentioning

confidence: 95%

ATP hydrolysis-dependent asymmetry of the conformation of CFTR channel pore

2011

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“…In the present work, we measured the mechanical properties of living BQ2 cells, a stably cystic fibrosis transmembrane conductance regulator [20,56] (CFTR)-overexpressing Chinese hamster ovary (CHO) cell line, as a function of the type and size of the colloidal probe. To analyze the acquired data, we calculated the Young's modulus of the cells by an implementation of the Hertz model.…”

Section: Introductionmentioning

confidence: 99%

Elasticity measurement of living cells with an atomic force microscope: data acquisition and processing

Carl

Schillers

2008

Pflugers Arch - Eur J Physiol

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201

176

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Elasticity of living cells is a parameter of increasing importance in cellular physiology, and the atomic force microscope is a suitable instrument to quantitatively measure it. The principle of an elasticity measurement is to physically indent a cell with a probe, to measure the applied force, and to process this force-indentation data using an appropriate model. It is crucial to know what extent the geometry of the indenting probe influences the result. Therefore, we indented living Chinese hamster ovary cells at 37 degrees C with sharp tips and colloidal probes (spherical particle tips) of different sizes and materials. We furthermore developed an implementation of the Hertz model, which simplifies the data processing. Our results show (a) that the size of the colloidal probe does not influence the result over a wide range (radii 0.5-26 microm) and (b) indenting cells with sharp tips results in higher Young's moduli ( approximately 1,300 Pa) than using colloidal probes ( approximately 400 Pa).

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“…The low [BPG] in the patient with the G551D mutation possibly results from changed enzyme activities because no differences in erythrocyte physiology were detectable. CFTR is incorporated into the red cell membrane (e. g. [53]), but a relation to BPG metabolism remains speculative.…”

Section: Discussionmentioning

confidence: 99%

Hemoglobin Oxygen Affinity in Patients with Cystic Fibrosis

et al. 2014

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In patients with cystic fibrosis lung damages cause arterial hypoxia. As a typical compensatory reaction one might expect changes in oxygen affinity of hemoglobin. Therefore position (standard half saturation pressure P50st) and slope (Hill’s n) of the O2 dissociation curve as well as the Bohr coefficients (BC) for CO2 and lactic acid were determined in blood of 14 adult patients (8 males, 6 females) and 14 healthy controls (6 males, 8 females). While Hill’s n amounted to approximately 2.6 in all subjects, P50st was slightly increased by 1mmHg in both patient groups (controls male 26.7±0.2, controls female 27.0±0.1, patients male 27.7±0.5, patients female 28.0±0.3 mmHg; mean and standard error, overall p<0.01). Main cause was a rise of 1–2 µmol/g hemoglobin in erythrocytic 2,3-biphosphoglycerate concentration. One patient only, clearly identified as an outlier and with the mutation G551D, showed a reduction of both P50st (24.5 mmHg) and [2,3-biphosphoglycerate] (9.8 µmol/g hemoglobin). There were no differences in BCCO2, but small sex differences in the BC for lactic acid in the controls which were not detectable in the patients. Causes for the right shift of the O2 dissociation curve might be hypoxic stimulation of erythrocytic glycolysis and an increased red cell turnover both causing increased [2,3-biphosphoglycerate]. However, for situations with additional hypercapnia as observed in exercising patients a left shift seems to be a more favourable adaptation in cystic fibrosis. Additionally when in vivo PO2 values were corrected to the standard conditions they mostly lay left of the in vitro O2 dissociation curve in both patients and controls. This hints to unknown fugitive factors influencing oxygen affinity.

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Imaging CFTR in its native environment

Cited by 28 publications

References 114 publications

ATP hydrolysis-dependent asymmetry of the conformation of CFTR channel pore

ATP hydrolysis-dependent asymmetry of the conformation of CFTR channel pore

Elasticity measurement of living cells with an atomic force microscope: data acquisition and processing

Hemoglobin Oxygen Affinity in Patients with Cystic Fibrosis

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