Apparent affinity of CFTR for ATP is increased by continuous kinase activity

AbstractThe cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel which is activated by protein phosphorylation and nucleoside triphosphates. We demonstrate here that fusion of the soluble catalytic subunit of cAMP-dependent protein kinase to the membrane protein bacteriorhodopsin yields a constitutively active protein kinase which activates CFTR effectively. As it is membrane-bound it is particularly useful for continuous perfusion of excised inside-out patches. We also tested the effect of a naturally membrane-bound protein kinase, cGMP-dependent protein kinase II, on CFTR. Both kinases, when continuously active, increase apparent affinity of CFTR to ATP about two-fold emphasizing the role of phosphorylation in modulating the interaction of ATP with the nucleotide binding domains

SIPcharts using uniform ultra-thin and thin fluorescent layers for z-response measurements in two-photon excitation fluorescence microscopy

Layer-by-Layer or self-assembly techniques can be used to prepare fluorescent polymer samples on glass coverslips serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general for sectioning microscopy.1-3 Layers can be realized as ultra-thin ( 6a 100 nm) or thin (approx. 100 nm) characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized in a Sectioned Imaging property chart (SIPchart),3 we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the zresponse of the system over a full field of view area. These samples are also useful for monitoring photobleaching behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be efficiently used for real time alignment of the microscope

SIPcharts using uniform ultra-thin and thin layers for Z-response measurements in two-photon excitation fluorescence microscopy

Layer-by-Layer or self-assembly techniques can be used to prepare fluorescent polymer samples on glass coverslips serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general for sectioning microscopy.1-3 Layers can be realized as ultra-thin (≪ 100 nm) or thin (approx. 100 nm) characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized in a Sectioned Imaging property chart (SIPchart),3 we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the zresponse of the system over a full field of view area. These samples are also useful for monitoring photobleaching behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be efficiently used for real time alignment of the microscope