161 research outputs found
Muscarine and t-LHRH suppress M-current by activating an IAP- insensitive G-protein
No full textThe control of M-current by muscarinic ACh receptors and luteinizing hormone releasing hormone (LHRH) receptors was studied in dialyzed frog sympathetic ganglion neurons. M-current was recorded in dialyzed cells without run-down or changes in its biophysical properties and could be reversibly suppressed by muscarine and teleost LHRH (t-LHRH). However, dialysis with internal solutions lacking ATP or substituting with APP(NH)P caused the loss of M-current, suggesting that dephosphorylation suppresses the activity of M-channels. M-current over- recovers after agonist addition and removal to a size 30% larger than control, as if latent channels are activated during the recovery. Dialysis of cells with the G-protein activators GTP gamma S, fluoride, and aluminum fluoride causes loss of M-current. G-protein activation by receptors was confirmed by dialysis with low concentrations of GTP gamma S in competition with GTP. This prevents the rapid loss of M- current, but addition of muscarine or t-LHRH caused irreversible loss of M-current, suggesting that both transmitter receptors do suppress M- current by activating a G-protein. Suppression of M-current was not affected by treatment with 0.1 microgram/ml pertussis toxin (IAP) for 24–48 hr. In addition, based on the lack of IAP-specific labeling of frog sympathetic neuron membrane proteins, no IAP-sensitive G-proteins are present in these cells. These results indicate that an IAP- insensitive G-protein couples muscarinic and LHRH receptors to the suppression of M-current.</jats:p
A conserved pre-block interaction motif regulates potassium channel activation and N-type inactivation.
No full textN-type inactivation occurs when the N-terminus of a potassium channel binds into the open pore of the channel. This study examined the relationship between activation and steady state inactivation for mutations affecting the N-type inactivation properties of the Aplysia potassium channel AKv1 expressed in Xenopus oocytes. The results show that the traditional single-step model for N-type inactivation fails to properly account for the observed relationship between steady state channel activation and inactivation curves. We find that the midpoint of the steady state inactivation curve depends in part on a secondary interaction between the channel core and a region of the N-terminus just proximal to the pore blocking peptide that we call the Inactivation Proximal (IP) region. The IP interaction with the channel core produces a negative shift in the activation and inactivation curves, without blocking the pore. A tripeptide motif in the IP region was identified in a large number of different N-type inactivation domains most likely reflecting convergent evolution in addition to direct descent. Point mutating a conserved hydrophobic residue in this motif eliminates the gating voltage shift, accelerates recovery from inactivation and decreases the amount of pore block produced during inactivation. The IP interaction we have identified likely stabilizes the open state and positions the pore blocking region of the N-terminus at the internal opening to the transmembrane pore by forming a Pre-Block (P state) interaction with residues lining the side window vestibule of the channel
Transformation of Functional Programs into Data Flow Graphs implemented with PVM
No full text. We present an implementation of the functional language FASAN for automatic coarse-grain program parallelization on workstation clusters. It is designed primarily for recursive numerical algorithms with distributed tree-like data structures and it exploits the maximal inherent parallelism of a program. Based on the stream and data flow semantics of the language, the compiler generates C procedures for building the data flow graph as dynamic data structure. FASAN schedulers evaluate the function nodes in parallel, and provide for all necessary communication using the PVM library. The new concept of "wrapper streams" for tree data structures avoids superfluous synchronization. 1 Introduction Modern numerical algorithms concerning partial differential equations are often based on the technique of recursive domain decomposition or sub-structuring [2]. Instead of arrays, trees are the natural and suitable data structures for implementations of those recursive algorithms. However, their p..
Development and optimization of photopolymerizable slurries for the Lithography-based Ceramic Manufacturing process
No full textDie vorliegende Arbeit beschäftigt sich mit den, für den Lithography-based Ceramic Manufacturing (LCM) Prozess als Ausgangsmaterial dienenden, photopolymerisierbaren keramischen Schlickersystemen. Diese stellen kolloidale Suspensionen dar. Keramische Partikel sind mithilfe eines Dispergieradditivs in einem organischen Gemisch aus Lösungsmittel und Monomeren fein dispergiert. Ein Photoinitiator ermöglicht durch selektive Belichtung des Schlickers das schichtweise Aushärten und schlussendliche Strukturieren dreidimensionaler Formkörper. In einer abschließenden thermischen Behandlung wird die organische Matrix der Bauteile ausgebrannt und die Keramikpartikel zu einer dichten Keramik ( 99 % theor. Dichte) gesintert. Bauteile hergestellt im LCM Prozess zeichnen sich durch hohe Auflösung ( 99 % theor. density). Parts manufactured in the LCM process excel in high resolution (< 25 µm) as well as high surface quality. Slurry systems are covered for the dental ceramics zirconia, an oxide ceramic with good mechanical properties used for restorations in the posterior tooth region and lithium disilicate, a glass ceramic with tooth-like optical properties (translucency, color) used for highly aesthetic restorations in the anterior region. Furthermore, suspensions for the bioceramic tricalcium phosphate are examined. There are three main requirements to ceramic-filled slurries in the LCM process, long-time stability against sedimentation, stability against separation due to occuring shear forces, and an easy, time efficient debinding of the manufactured green bodies. For this purpose different approaches are pursued to enable a stable, flawless and reproducible production of parts out of high-performance ceramics. Through applying a thermoplastic component in existing slurry formulations instead of the diluent or by using fumed silica, an inorganic rheology additive, the ceramic slurries could be stabilized against sedimentation. However, during the layer-wise manufacturing of parts difficulties arose due to separation processes or deficient flow properties. Stereolithography as an optical forming process for the production of high quality parts requires specific optical properties of the raw material. On this account slurry systems with improved optical properties (matching of the refractive index of organic components and ceramic powder) are developed which allow the manufacturing of parts with the highest possible resolution and surface quality (minimal wall thickness of 100 µm). Due to the modified organic fraction the slurries show thixotropic flow behavior. Thereby long-time stability against sedimentation is achieved. Besides the slurry development an adaptation of the overall process chain, including additive manufacturing of parts, thermal debinding of green bodies and final sintering was necessary. Additionally, with this slurry system the author could establish a time efficient debinding process (dental crown in < 3 h)
Slowed Closing produced by N-type Inactivation matches the voltage-dependence for normal channel closing.
No full text<p>A) Time course for AKv1 tail currents matches the Inactivation recovery time course. B) Kinetics for normal closing measured in AKv1(Δ2-57) and recovery tails measured in AKv1 are voltage dependent. C) Comparing tail current closing rates from single exponential fits. Voltage-dependence for AKv1(Δ2-57) closing matches the voltage-dependence for AKv1 tail currents despite the dramatically different rates. D) Ratio of tail closing rates predicts a consistent value for K<sub>I</sub>. For wild type AKv1 this value is similar to K<sub>I</sub><sup>1</sup> measured from the fraction of current that is not inactivated at the end of a pulse.</p
<i>Drosophila Shaker</i> based Model Poorly Predicts AKv1 Steady State Inactivation.
No full text<p>Best fit to AKv1(E2x) channel data is shown in black. While there is a trend in the data matching the prediction of the model, error bars for most data points do not contact the best fit line. The y-intercept of the best fit is significantly different from the activation midpoint for AKv1(Δ2-57).</p
Modeling the Pre-Block Interaction.
No full text<p>A) Minimal AKv1 N-type Inactivation gating scheme. AKv1 gating model is only slightly more complicated than the Single Step <i>Drosophila</i> Model (red states) because in addition to the <b>C</b>, <b>O</b> and <b>I</b> labels to indicate the pore state, a subscript is needed to indicate : <b>P</b>- the <b>P</b> site bound states (black states) that shift activation and enhances inactivation and, <b>W</b>- a separate <b>W</b>ithheld state (blue state), from which the IB region cannot directly access the pore block state. Rate constants k<sub>c</sub>(v) = 40(−0.015) and k<sub>o</sub>(v) = 1500(0.32) as described previously. Equilibrium constants and cooperativity factor α determined as described in the text. The model produces a reasonable fit for the steady state properties for AKv1, AKv1(I8Q), and AKv1(E2D) (with K<sub>I</sub> changed to 0.1). Accurate activation and inactivation kinetics at strong depolarizations requires additional steps along the red pathway to rate limit the kinetics. If direct closing from O<sub>P</sub> and O<sub>W</sub> open states is included using the same k<sub>c</sub>(v) value then closing and recovery kinetics for AKv1, AKv1(I8Q), and AKv1(E2D) channels at strong negative potentials can be reproduced with this model if the K<sub>I</sub> equilibrium is made rapid. B) Structural model of key regions involved in N-type inactivation based on the 3LUT structure of Kv1.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079891#pone.0079891-Chen1" target="_blank">[23]</a>. A tilted perspective (see inset upper right) of the channel showing the inner aqueous volume of the channel in gray. Residue Tyr417 is shown in red and the S4-S5 linker in green. Selectivity filter is marked by the locations of the potassium ions in purple. C) Internal aqueous volume of the channel seen from a side perspective divided into the pore inner vestibule (white) and the side window vestibule (green). Volumes of these regions are given in the matching color. A single subunit P region backbone trace from the S4-S5 linker to the end of the determined S6 structure is shown along with its residue Tyr417 (red) and S4-S5 linker (brown) highlighted. The S4-S5 linker from the adjacent subunit is close to Tyr417 and therefore is also shown. D) Same picture only rotated 90<sup>o</sup> to show the locations of Tyr417 and the S4-S5 linkers from 2 subunits relative to the pore inner vestibule and the side window vestibule. Both Tyr417 and the S4-S5 linkers are side window vestibule lining residues, not pore inner vestibule lining residues.</p
Slowed Closing predicts steady state inactivation midpoints for AKv1(E2x) series mutants.
No full text<p>A) Best fit to AKv1(E2x) channel data is shown in black. Prediction for the steady state inactivation midpoints based on AKv1(Δ2-57) activation gating shown in orange. AKv1(E2x) data are well fit with a linear model (r = −0.99); however, the y-intercept is more negative than expected and the slope is flatter than predicted from AKv1(Δ2-57) activation. B) Predicted value for k<sub>s</sub> from the fit is smaller than the activation curve k<sub>s</sub>, and similar to k<sub>s</sub> values measured from the inactivation curves, as expected from the more complex multi-step activation of the real channel.</p
Identification the Inactivation Proximal (IP) Domain.
No full text<p>A) Representative currents for N-terminal deletions. Removal of the initial 5 residues eliminates N-type inactivation. B) Activation midpoint for AKv1(Δ2-5) is shifted to a more negative midpoint compared to larger N-terminal Deletions AKv1(Δ2-14) and AKv1(Δ2-57). C) Deletion effects on activation midpoint identify the IP region between residues 5-14. D) Despite the shift in activation midpoint, AKv1(Δ2-5) closing kinetics and voltage dependence are similar to AKv1(Δ2-57). D) Using AKv1(Δ2-5) activation midpoint and slope from AKv1 inactivation accurately predicts inactivation midpoints for AKv1(E2x) mutant series (Model Prediction line).</p
Identification of Conserved Motif in IP Domain.
No full text<p>A) IP Domain retained in AKv1(Δ2-5) and deleted in AKv1(Δ2-14) contains a highly conserved [(A/V)-(G/S/C)-(H<sub>5</sub>)] Motif. Mutations to residue Leu7 from <i>Drosophila</i> ShB channel, highlighted in red, that make this residue more polar disrupt ShB N-type inactivation. B) AKv1(Δ2-5, I8Q) mutant shows expected non-inactivating currents. C) Activation Curve for AKv1(Δ2-5, I8Q) is shifted back to more positive potentials and matches AKv1(Δ2-57). D) Summary data showing deletions and mutations identifying the IP Domain and the disruption of IP Domain effects on activation by the I8Q mutation.</p
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