Structure-function analyses of small-conductance, calcium-activated potassium channels

Ion channels are integral membrane proteins present in all cells. They are highly selective and assure a high rate for ions down their electrochemical gradient. In particular, small-conductance calcium-activated potassium channels (SK) are conducting potassium ions and are activated by binding of calcium ions to calmodulin, which is constitutively bound to the carboxy-terminus of each SK channel -subunit. Until now, only three SK channel subunits have been cloned, SK1, SK2 and SK3. Sequence alignment shows that the transmembrane and pore regions are highly conserved, while a high grade of divergence is observed in the amino- and carboxy-termini of the three subunits. In order to determine the expression of the different SK channel subtypes, pharmacological tols such as apamin and d-tubocurarine have been widely used. In this work, I show the characterization of a novel toxin, tamapin, isolated from the scorpion Mesobuths tamulus, which targets SK channels. Our experiments show that this toxin is more potent in blocking SK2 channels than apamin. Furthermore, tamapin only blocked the SK1 and SK3 channels at higher concentrations, with higher efficiency to block SK3 than SK1. Therefore, tamapin should be a good pharmacological tool to determine the molecular composition of native SK channels underlying calcium-activated potassium currents in various tissues. Secondly, I determined the molecular mechanism that prevents the formation of functional SK1 channels cloned from the rat brain (rSK1). Until now, little information was available on the rSK1 channels. rSK1 shows highly sequence identity (84%) with the human homologue, hSK1. hSK1 subunits form functional potassium channels that are blocked by apamin and d-tubocurarine. However, when I expressed rSK1 in HEK-293 cells no potassium currents above background were observed, although immunofluorescence experiments using a specific antibody against the rSK1 protein showed expression of the channel. I generated rSK1 core chimeras in which I exchanged the amino-and/carboxy-terminus with the same region of rSK2 or hSK1. Exchange of amino-and carboxy-terminus or only of the carboxy-terminus resulted in the formation of functional potassium channels. Furthermore, I used these functional chimeras to determine the toxin sensitivity of rSK1 for apamin and d-tubocurarine. Surprisingly, when these blockers wre applied, no sensitivity was observed, although hSK1 and rSK1 show a complete sequence identity in the pore region, which is suggested to contain the binding site for apamin. Finally, I characterized a novel splice variant of the calcium-activated potassium channel subunit rSK2, referred to as rSK2-860. The rSK2-860 cDNA codes for a protein which is 275 amino acids longer at the amino-terminus when compared with originally cloned rSK2 subunit. Transfection of rSK2-860 in different cell lines resulted in a surprising expression pattern of the protein. Th protein formed small clusters around the cell nucleus, but no membrane stain could be observed. This data shows that the additional 275 amino acid-long stretch at the amino-terminus is responsible for retention and clustering of rSK2-860 protein. In order to narrow down the region responsible for this phenotype, I generated truncated proteins. This resulted in the isolation of an 100 amino acid-long region that seems to be responsible for the retention and clustering of rSK2-860 channels. Further truncations and deletions could help us to find the exact signal which is responsible for this characteristic behavior of the rSK2-860 protein

Structure-function analyses of small-conductance, calcium-activated potassium channels

Ion channels are integral membrane proteins present in all cells. They are highly selective and assure a high rate for ions down their electrochemical gradient. In particular, small-conductance calcium-activated potassium channels (SK) are conducting potassium ions and are activated by binding of calcium ions to calmodulin, which is constitutively bound to the carboxy-terminus of each SK channel -subunit. Until now, only three SK channel subunits have been cloned, SK1, SK2 and SK3. Sequence alignment shows that the transmembrane and pore regions are highly conserved, while a high grade of divergence is observed in the amino- and carboxy-termini of the three subunits. In order to determine the expression of the different SK channel subtypes, pharmacological tols such as apamin and d-tubocurarine have been widely used. In this work, I show the characterization of a novel toxin, tamapin, isolated from the scorpion Mesobuths tamulus, which targets SK channels. Our experiments show that this toxin is more potent in blocking SK2 channels than apamin. Furthermore, tamapin only blocked the SK1 and SK3 channels at higher concentrations, with higher efficiency to block SK3 than SK1. Therefore, tamapin should be a good pharmacological tool to determine the molecular composition of native SK channels underlying calcium-activated potassium currents in various tissues. Secondly, I determined the molecular mechanism that prevents the formation of functional SK1 channels cloned from the rat brain (rSK1). Until now, little information was available on the rSK1 channels. rSK1 shows highly sequence identity (84%) with the human homologue, hSK1. hSK1 subunits form functional potassium channels that are blocked by apamin and d-tubocurarine. However, when I expressed rSK1 in HEK-293 cells no potassium currents above background were observed, although immunofluorescence experiments using a specific antibody against the rSK1 protein showed expression of the channel. I generated rSK1 core chimeras in which I exchanged the amino-and/carboxy-terminus with the same region of rSK2 or hSK1. Exchange of amino-and carboxy-terminus or only of the carboxy-terminus resulted in the formation of functional potassium channels. Furthermore, I used these functional chimeras to determine the toxin sensitivity of rSK1 for apamin and d-tubocurarine. Surprisingly, when these blockers wre applied, no sensitivity was observed, although hSK1 and rSK1 show a complete sequence identity in the pore region, which is suggested to contain the binding site for apamin. Finally, I characterized a novel splice variant of the calcium-activated potassium channel subunit rSK2, referred to as rSK2-860. The rSK2-860 cDNA codes for a protein which is 275 amino acids longer at the amino-terminus when compared with originally cloned rSK2 subunit. Transfection of rSK2-860 in different cell lines resulted in a surprising expression pattern of the protein. Th protein formed small clusters around the cell nucleus, but no membrane stain could be observed. This data shows that the additional 275 amino acid-long stretch at the amino-terminus is responsible for retention and clustering of rSK2-860 protein. In order to narrow down the region responsible for this phenotype, I generated truncated proteins. This resulted in the isolation of an 100 amino acid-long region that seems to be responsible for the retention and clustering of rSK2-860 channels. Further truncations and deletions could help us to find the exact signal which is responsible for this characteristic behavior of the rSK2-860 protein

A secondary resonance in Mercury's rotation

International audienceThe resonant rotation of Mercury can be modelised by a kernel model on which we can add perturbations. Our kernel model is a two-degree of freedom one written in Hamiltonian formalism. For this kernel, we consider that Mercury is solid and rotates on a Keplerian orbit. By introducing the perturbations due to the other planets of the Solar System, it appears that, in a particular case, our slow degree of freedom may enter into a 1:1 resonance with the Great Inequality of Jupiter and Saturn. Actually, as the moments of inertia of Mercury are still poorly known, this phenomenon cannot be excluded

Ink-jet printout of radiographs on transparent film and glossy paper versus monitor display: an ROC analysis

The aim of this study was to compare the depiction ability of small grayscale contrasts in ink-jet printouts of digital radiographs on different print media with CRT monitor. A CCD-based digital cephalometric image of a stepless aluminum wedge containing 50 bur holes of different depth was cut into 100 isometric images. Each image was printed on glossy paper and on transparent film by means of a high-resolution desktop inkjet printer at specific settings. The printed images were viewed under standardized conditions, and the perceptibility of the bur holes was evaluated and compared to the perceptibility on a 17-in CRT monitor. Thirty observers stated their blinded decision on a five-point confidence scale. Areas (Az) under receiver operating characteristics curves were calculated and compared using the pair wise sign tests. Overall agreement was estimated using Cohen's kappa device and observer bias using McNemar's test. Glossy paper prints and monitor display revealed significantly higher (P < 0.001) average Az values (0.83) compared to prints on transparent film (0.79), which was caused by higher sensitivity. Specificity was similar for all modalities. The sensitivity was dependent on the mean gray scale values for the transparent fil

The influence of bone substitute materials on the bone volume after maxillary sinus augmentation: a microcomputerized tomography study

Objectives: This study aims to evaluate the effect of adding bone substitute materials (BSM) to particulated autogenous bone (PAB) on the volume fraction (Vf) of newly formed bone after maxillary sinus augmentation. Materials and methods: Thirty healthy patients undergoing maxillary sinus augmentation were included. PAB (N = 10), mixtures of PAB and beta-tricalciumphosphate (PAB/β-TCP) (N = 10), as well as PAB and β-TCP and hydroxyapatite (PAB/HA/β-TCP) (N = 10) were randomly used for sinus augmentation. A sample of the graft material was maintained from each patient at time of maxillary sinus augmentation, and Vfs of the PAB and/or BSM in the samples were determined by means of microcomputerized tomography (μ-CT). Five months later, samples of the grafted areas were harvested during implantation using a trephine bur. μ-CT analysis of these samples was performed, and the Vf of bone and BSM were compared with the data obtained 5months earlier from the original material. Results: The mean Vf of the bone showed a statistically significant increase (p < 0.05) in all groups after a healing period of 5months without statistically significant difference between the groups. Conclusions: With regard to the increase of bone volume, it is not relevant if PAB is used alone or combined with β-TCP or HA/β-TCP. Clinical relevance: The amount of PAB and associated donor site morbidity may be reduced by adding BSM for maxillary sinus augmentatio

On the oscillations in Mercury's obliquity

One major objective of MESSENGER and BepiColombo spatial missions is to accurately measure Mercury's rotation and its obliquity in order to obtain constraints on internal structure of the planet. Which is the obliquity's dynamical behavior deriving from a complete spin-orbit motion of Mercury simultaneously integrated with planetary interactions? We have used our SONYR model integrating the spin-orbit N-body problem applied to the solar System (Sun and planets). For lack of current accurate observations or ephemerides of Mercury's rotation, and therefore for lack of valid initial conditions for a numerical integration, we have built an original method for finding the libration center of the spin-orbit system and, as a consequence, for avoiding arbitrary amplitudes in librations of the spin-orbit motion as well as in Mercury's obliquity. The method has been carried out in two cases: (1) the spin-orbit motion of Mercury in the 2-body problem case (Sun-Mercury) where an uniform precession of the Keplerian orbital plane is kinematically added at a fixed inclination (S2K case), (2) the spin-orbit motion of Mercury in the N-body problem case (Sun and planets) (Sn case). We find that the remaining amplitude of the oscillations in the Sn case is one order of magnitude larger than in the S2K case, namely 4 versus 0.4 arcseconds (peak-to-peak). The mean obliquity is also larger, namely 1.98 versus 1.80 arcminutes, for a difference of 10.8 arcseconds. These theoretical results are in a good agreement with recent radar observations but it is not excluded that it should be possible to push farther the convergence process by drawing nearer still more precisely to the libration center.Comment: 30 pages, 3 tables, 8 figures, accepted to Icarus (26 Jul 2007