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Kaliumionen (K<sup>+) sind die am häufigsten vorkommenden anorganischen Kationen in Pflanzen. Gemessen am Trockengewicht kann ihr Anteil bis zu 10% ausmachen. Kaliumionen übernehmen wichtige Funktionen in verschiedenen Prozessen in der Pflanze. So sind sie z.B. essentiell für das Wachstum und für den Stoffwechsel. Viele wichtige Enzyme arbeiten optimal bei einer K<sup>+ Konzentration im Bereich von 100 mM. Aus diesem Grund halten Pflanzenzellen in ihren Kompartimenten, die am Stoffwechsel beteiligt sind, eine kontrollierte Kaliumkonzentration von etwa 100 mM aufrecht. Die Aufnahme von Kaliumionen aus dem Erdreich und deren Transport innerhalb der Pflanze und innerhalb einer Pflanzenzelle wird durch verschiedene Kaliumtransportproteine ermöglicht. Die Aufrechterhaltung einer stabilen K<sup>+ Konzentration ist jedoch nur möglich, wenn die Aktivität dieser Transportproteine einer strikten Kontrolle unterliegt. Die Prozesse, die die Transportproteine regulieren, sind bis heute nur ansatzweise verstanden. Detailliertere Kenntnisse auf diesem Gebiet sind aber von zentraler Bedeutung für das Verständnis der Integration der Transportproteine in das komplexe System des pflanzlichen Organismus. In dieser Habilitationsschrift werden eigene Publikationen zusammenfassend dargestellt, in denen die Untersuchungen verschiedener Regulationsmechanismen pflanzlicher Kaliumkanäle beschrieben werden. Diese Untersuchungen umfassen ein Spektrum aus verschiedenen proteinbiochemischen, biophysikalischen und pflanzenphysiologischen Analysen. Um die Regulationsmechanismen grundlegend zu verstehen, werden zum einen ihre strukturellen und molekularen Besonderheiten untersucht. Zum anderen werden die biophysikalischen und reaktionskinetischen Zusammenhänge der Regulationsmechanismen analysiert. Die gewonnenen Erkenntnisse erlauben eine neue, detailliertere Interpretation der physiologischen Rolle der Kaliumtransportproteine in der Pflanze.
Among all voltage-gated K+ channels from the model plant Arabidopsis thaliana, the weakly rectifying K+ channel (K-weak channel) AKT2 displays unique gating properties. AKT2 is exceptionally regulated by phosphorylation: when nonphosphorylated AKT2 behaves as an inward-rectifying potassium channel; phosphorylation of AKT2 abolishes inward rectification by shifting its activation threshold far positive (>200 mV) so that it closes only at voltages positive of + 100 mV. In its phosphorylated form, AKT2 is thus locked in the open state in the entire physiological voltage range. To understand the molecular grounds of this unique gating behavior, we generated chimeras between AKT2 and the conventional inward-rectifying channel KAT1. The transfer of the pore from KAT1 to AKT2 altered the permeation properties of the channel. However, the gating properties were unaffected, suggesting that the pore region of AKT2 is not responsible for the unique K-weak gating. Instead, a lysine residue in S4, highly conserved among all K-weak channels but absent from other plant K+ channels, was pinpointed in a site-directed mutagenesis approach. Substitution of the lysine by serine or aspartate abolished the "open-lock" characteristic and converted AKT2 into an inward- rectifying channel. Interestingly, phosphoregulation of the mutant AKT2-K197S appeared to be similar to that of the K-in channel KAT1: as suggested by mimicking the phosphorylated and dephosphorylated states, phosphorylation induced a shift of the activation threshold of AKT2-K197S by about +50 mV. We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation. The phosphorylation-induced reduction of the activation energy in AKT2 is similar to 6 kT larger than in the K197S mutant. It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it
Members of the Shaker-like plant K+ channel family share a common structure, but are highly diverse in their function: they behave as either hyperpolarization-activated inward-rectifying (K-in) channels, or leak-like (K-weak) channels, or depolarization-activated outward-rectifying (K-out) channels. Here we created 256 chimeras between the K-in channel KAT1 and the K-out channel SKOR. The chimeras were screened in a potassium-uptake deficient yeast strain to identify those, which mediate potassium inward currents, i.e., which are functionally equivalent to KAT1. This strategy allowed Lis to identify three chimeras which differ from KAT1 in three parts of the polypeptide: the cytosolic N- terminus, the cytosolic C-terminus, and the putative voltage-sensor S4. Additionally, mutations in the K-out Channel SKOR were generated in order to localize molecular entities underlying its depolarization activation. The triple mutant SKOR-D312N-M313L-1314G, carrying amino-acid changes in the S6 segment, was identified as a channel which did not display any rectification in the tested voltage-range. (C) 2005 Elsevier Inc. All rights reserved
Expressed sequence tags (ESTs) represent a huge resource for the discovery of previously unknown genetic information and functional genome assignment. In this study we screened a collection of 178 292 ESTs from Arabidopsis thaliana by testing them against previously annotated genes of the Arabidopsis genome. We identified several hundreds of new transcripts that match the Arabidopsis genome at so far unassigned loci. The transcriptional activity of these loci was independently confirmed by comparison with the Salk Whole Genome Array Data. To a large extent, the newly identified transcriptionally active genomic regions do not encode 'classic' proteins, but instead generate non-coding RNAs and/or small peptide-coding RNAs of presently unknown biological function. More than 560 transcripts identified in this study are not represented by the Affymetrix GeneChip arrays currently widely used for expression profiling in A. thaliana. Our data strongly support the hypothesis that numerous previously unknown genes exist in the Arabidopsis genome