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Plasmakatalyse op nanoschaal: Modelontwikkeling voor diffusie van plasmadeeltjes in poriën en studie van het katalytisch gedrag aan het porie-oppervlak.
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Plasma catalysis at the nanoscale: Model development for diffusion of plasma particles in pores and study of the catalytic behaviour at the pore surface.
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De specifieke doelstellingen zijn: (1) Cloneren van nieuwe isovormen van de HCN-familie en testen voor het bestaan van alternatieve splice varianten en geassocieerde beta-subunits; (2) Testen of het GYG-motief in de porie kritisch is voor de selectiviteit en de permeatie-eigenschappen van HCN-kanalen; (3) Testen of het S4-segment een uitwaarts bewegende voltage sensor is en hoe deze kanaalopening veroorzaakt bij hyperpolarizatie.
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The rhythmic activity of the heart originates in the sino-atrial node. Pacemaking cells display a slow diastolic depolarization which is controlled to a large extent by a mixed Na/K current. In contrast to most voltage-gated channels, this current activates slowly upon hyperpolarization. Therefore the current was named If (f for funny) or Ih (h for hyperpolarization-activated). A total of four subunits have already been cloned for these hyperpolarization-activated & cyclic nucleotide gated (HCN) channels. Their molecular structure displays remarkable similarities with highly-selective K channels that open upon depolarization. The specific aims of this proposal are to: (1) Clone novel isoforms of the HCN family and test for the existence of alternatively spliced variants or beta-subunits; (2)Test whether the GYG motif in the pore segment is critical for the (cation) selectivity and permeation properties of HCN channels; (3) Test whether S4 moves outwardly as in Shaker channels but couples differently to the activation gate.
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Bij spanningsafhankelijk ionenkanalen wordt door de beweging van S4 (spanningssensor) een soort poort in de kanaalporie (S6) geopend (of gesloten). De porie is tevens de bindingsplaats voor verscheidene farmaca.
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Ion channels are transmembrane proteins that allow selective ion permeation through a central pore which opens and closes depending on an external stimulus. In the case of voltage dependent ion channels it is the movement of S4 ' the voltage sensor ' that triggers the opening of the gate in the channel pore (mainly S6). Specific aims of this project are (1) to test that specific pore lining residues in the S6 segment are involved not only in drug binding but also in channel activation, (2) to distinguish between molecular determinants for channel block and agonist effects of fatty acids and antiarrhythmic drugs, and (3) to evaluate whether stable cell lines expressing specific channel subunits can be used in high throughput screening assays for antiarrhythmic drugs or as an in vitro system to assess the anti- or pro-arrhythmic potential of various drugs (antihistaminics, antibiotics). This will be studied using biophysical analysis of ion channel gating using patch clamp techniques in combination with molecular manipulation of the channel structure such as channel chimeras, site-directed mutagenesis, cysteine scanning and site directed fluorescence. The specific clones employed in this study will be hKv1.5, Kv4.2, HERG and KvLQT1 which are major components of repolarizing K currents in cardiac tissue.
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Bij spanningsafhankelijk ionenkanalen wordt door de beweging van S4 (spanningssensor) een soort poort in de kanaalporie (S6) geopend (of gesloten). De porie is tevens de bindingsplaats voor verscheidene farmaca.
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Ion channels are transmembrane proteins that allow selective ion permeation through a central pore which opens and closes depending on an external stimulus. In the case of voltage dependent ion channels it is the movement of S4 ' the voltage sensor ' that triggers the opening of the gate in the channel pore (mainly S6). Specific aims of this project are (1) to test that specific pore lining residues in the S6 segment are involved not only in drug binding but also in channel activation, (2) to distinguish between molecular determinants for channel block and agonist effects of fatty acids and antiarrhythmic drugs, and (3) to evaluate whether stable cell lines expressing specific channel subunits can be used in high throughput screening assays for antiarrhythmic drugs or as an in vitro system to assess the anti- or pro-arrhythmic potential of various drugs (antihistaminics, antibiotics). This will be studied using biophysical analysis of ion channel gating using patch clamp techniques in combination with molecular manipulation of the channel structure such as channel chimeras, site-directed mutagenesis, cysteine scanning and site directed fluorescence. The specific clones employed in this study will be hKv1.5, Kv4.2, HERG and KvLQT1 which are major components of repolarizing K currents in cardiac tissue.