On the problem of supersonic gas flow in two-dimensional channel with the oscillating upper wall

In the present paper we solve the problem of supersonic gas flow in two-dimensional channel with the moving upper wall making oscillations according to the harmonic law. In order to get a numerical solution for gas dynamics equations we have implemented a difference scheme with space and time approximation of the first order and one with space approximation of the second order. Depending on a type of harmonic law and initial gas inflow conditions, the peculiarities of angle-shock wave propagation in moving curvilinear domains have been investigated. It has been determined that the increase of oscillation amplitude causes the increase of shock wave intensity. It has been shown that under particular oscillation amplitude the moving wall has practically no effect on the flow within the domain

Der spannungsgesteuerte Chloridkanal CIC-2 und seine Bedeutung in der Pathogenese von idiopathischer generalisierter Epilepsie

ClC-2 is a member of the ClC-family of voltage-gated chloride channels. Within this family, ClC-2 is unique in being activated by hyperpolarization, low extracellular pH and hypotonic cell swelling. In this study, several biophysical properties of the human ClC-2 channel have been functionally characterized for the first time and compared with the corresponding properties of the rat isoform. Both proteins were expressed heterologously in tsA201-cells and were analyzed electrophysiologically using the whole-cell patch-clamp technique. hClC-2 and rClC-2 hardly differ in their activation properties. Both channels are virtually closed at positive potentials and activate slowly upon hyperpolarization. However, the human ClC-2 displays slower activation and deactivation kinetics than the rat isoform. A formerly hypothesized function as an inactivation domain for the cytoplasmatic N-terminus of ClC-2 could not be verified. A corresponding N-terminal deletion mutant exhibits a nearly unchanged gating behaviour which argues against the proposed inactivation model for this channel. However, the changed kinetics of the mutant imply an important role of the N-terminus in gating of ClC-2. Furthermore, it could be shown that the current block by external iodide is dependent on the membrane potential as well as on the conformational state of the channel. This argues against the existence of an intracellular inactivation domain as well. A dependence of the activation behaviour of ClC-2 on the transmembrane chloride gradient could be proven: the channel activates only at potentials negative to the reversal potential and therefore constitutes an exclusive efflux pathway for chloride ions. Based on this property, a model for the role of ClC-2 in neuronal chloride homeostasis has been proposed assigning the channel an important function in establishing and maintaining a low intracellular chloride concentration. In the second part of this work, three mutations of ClC-2 were studied that have recently been identified in patients with idiopathic generalized epilepsy (IGE). Two of these mutants, M200fsX231 and del74-117, were non-functional, neither as homodimers nor as heterodimers with one wildtype subunit. A correct trafficking of the mutated proteins to the cell membrane was demonstrated by fluorometric experiments and could therefore be discarded as possible cause for the dominant-negative effect of these mutations. Whether the lack of functionality is due to the missing ability of the mutated subunits to interact with wildtype subunits could not be shown unambiguously in FRET experiments. The loss of ClC-2 function in patients carrying these mutations might lead to an intracellular chloride accumulation that could result in an excitatory GABAergic response and neuronal hyperexcitability and thus represent a possible explanation for the epileptic phenotype. The third IGE mutation, G715E, does not impair the functionality of ClC-2 but leads to an altered voltage-dependence of channel gating: mutated channels open already at less negative potentials than the wildtype isoform. Under certain conditions, for example during the repolarization phase after intense synaptic activity, they conduct an increased outward chlorid current possibly leading to recurrent membrane depolarization and repeated action potentials. The G715E mutation therefore seems to cause epileptic seizures by a distinct mechanism comprising a gain-of-function of ClC-2. The results of this thesis demonstrate the importance of gating in ClC-2 for its function and establish CLCN2 as a disease-causing gene in idiopathic epilepsies

Der spannungsgesteuerte Chloridkanal CIC-2 und seine Bedeutung in der Pathogenese von idiopathischer generalisierter Epilepsie

ClC-2 is a member of the ClC-family of voltage-gated chloride channels. Within this family, ClC-2 is unique in being activated by hyperpolarization, low extracellular pH and hypotonic cell swelling. In this study, several biophysical properties of the human ClC-2 channel have been functionally characterized for the first time and compared with the corresponding properties of the rat isoform. Both proteins were expressed heterologously in tsA201-cells and were analyzed electrophysiologically using the whole-cell patch-clamp technique. hClC-2 and rClC-2 hardly differ in their activation properties. Both channels are virtually closed at positive potentials and activate slowly upon hyperpolarization. However, the human ClC-2 displays slower activation and deactivation kinetics than the rat isoform. A formerly hypothesized function as an inactivation domain for the cytoplasmatic N-terminus of ClC-2 could not be verified. A corresponding N-terminal deletion mutant exhibits a nearly unchanged gating behaviour which argues against the proposed inactivation model for this channel. However, the changed kinetics of the mutant imply an important role of the N-terminus in gating of ClC-2. Furthermore, it could be shown that the current block by external iodide is dependent on the membrane potential as well as on the conformational state of the channel. This argues against the existence of an intracellular inactivation domain as well. A dependence of the activation behaviour of ClC-2 on the transmembrane chloride gradient could be proven: the channel activates only at potentials negative to the reversal potential and therefore constitutes an exclusive efflux pathway for chloride ions. Based on this property, a model for the role of ClC-2 in neuronal chloride homeostasis has been proposed assigning the channel an important function in establishing and maintaining a low intracellular chloride concentration. In the second part of this work, three mutations of ClC-2 were studied that have recently been identified in patients with idiopathic generalized epilepsy (IGE). Two of these mutants, M200fsX231 and del74-117, were non-functional, neither as homodimers nor as heterodimers with one wildtype subunit. A correct trafficking of the mutated proteins to the cell membrane was demonstrated by fluorometric experiments and could therefore be discarded as possible cause for the dominant-negative effect of these mutations. Whether the lack of functionality is due to the missing ability of the mutated subunits to interact with wildtype subunits could not be shown unambiguously in FRET experiments. The loss of ClC-2 function in patients carrying these mutations might lead to an intracellular chloride accumulation that could result in an excitatory GABAergic response and neuronal hyperexcitability and thus represent a possible explanation for the epileptic phenotype. The third IGE mutation, G715E, does not impair the functionality of ClC-2 but leads to an altered voltage-dependence of channel gating: mutated channels open already at less negative potentials than the wildtype isoform. Under certain conditions, for example during the repolarization phase after intense synaptic activity, they conduct an increased outward chlorid current possibly leading to recurrent membrane depolarization and repeated action potentials. The G715E mutation therefore seems to cause epileptic seizures by a distinct mechanism comprising a gain-of-function of ClC-2. The results of this thesis demonstrate the importance of gating in ClC-2 for its function and establish CLCN2 as a disease-causing gene in idiopathic epilepsies

Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes

TRPM2 (transient receptor potential melastatin 2) is a Ca(2+)-permeable cation channel gated by ADPR (ADP-ribose) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC method to determine ADPR contents in HClO(4) cell extracts. The reversed-phase ion-pair HPLC method with an Mg(2+)-containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were approx. 5 μM in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP (N-formylmethionyl-leucylphenylalanine). We furthermore determined intracellular concentrations of cADPR (cyclic ADPR) with a cyclase assay involving enzymatic conversion of cADPR into NAD(+) and fluorimetric determination of NAD(+). Intracellular cADPR concentrations were approx. 0.2 μM and not altered by fMLP. In patch–clamp experiments, ADPR (0.1–100 μM) was dialysed into granulocytes to analyse its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (<10 nM) or a high (1 μM) intracellular Ca(2+) concentration. TRPM2 currents were significantly larger at high than at low [Ca(2+)] (e.g. −225±27.1 versus −7±2.0 pA/pF at 5 μM ADPR), but no currents at all were observed in the absence of ADPR (ADPR concentration ≤0.3 μM). cADPR (0.1, 0.3 and 10 μM) was without effect even in the presence of subthreshold ADPR (0.1 μM). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca(2+). Thus ADPR and Ca(2+) in concert behave as a messenger system for agonist-induced influx of Ca(2+) through TRPM2 in granulocytes