The Ca2+ -signaling pathway is an important mechanism of cell signaling. Ca2+ /Cal- modulin (CaM)-dependent protein kinases (CaMKs) are members of Ser/Thr protein kinase family. CaMKs are regulated by Ca2+ /CaM binding in response to increase in intracellular level of Ca2+ . An important member of this protein family is Ca2+ /CaM- dependent protein kinase kinase (CaMKK), which is an upstream activator of CaMKI and CaMKIV. There are two isoforms of CaMKK, CaMKK1 and CaMKK2. CaMKK1 is regulated not only by Ca2+ /CaM-binding, but also by phosphorylation by cAMP-dependent protein kinase A (PKA). PKA phosphorylation induces inter- action with the 14-3-3 proteins. Previous studies of interaction between CaMKK1 and 14-3-3 proteins suggested, that the interaction with 14-3-3 proteins keeps CaMKK1 in the PKA-induced inhibited state and blocks its active site. However, the exact mecha- nism of this inhibition is still unclear mainly due to the absence of structural data. Main aim of this diploma thesis was to characterize the protein complexes between CaMKK1, Ca2+ /CaM and 14-3-3γ using analytical ultracentrifugation, small angle X-ray scattering, and chemical cross-linking coupled to mass spectrometry. Analytical ultracentrifugation revealed concentration-dependent dimerization of CaMKK1, which is...

Im Rahmen der vorliegenden Arbeit wurde eine mögliche Beteiligung der Protein-Translokase des Endoplasmatischen Retikulums aus Canis familiaris an der Vermittlung eines passiven Calcium-Ausstromes aus dem ER-Lumen untersucht. Der Sec61-Komplex konstituiert eine ionenpermeable Pore im Translokon des Endoplasmatischen Retikulums. Der Kanal zeigt eine hohe Dynamik im Schaltverhalten mit einer Vielzahl von Unterleitwerten, deren Mittelwerte gut mit publizierten Daten übereinstimmen. Zudem besitzt die Pore eine geringe Anionenselektivität in Experimenten mit KCl-Lösungen. Unter Verwendung von CaCl2- und MgCl2-Elektrolyten steigt diese deutlich an, was in vivo den Sec61-vermittelten, passiven Calcium-Ausstrom aus dem ER limitieren kann. Calmodulin (CaM) konnte im Rahmen der vorliegenden Arbeit als potenter Effektor des Sec61-Kanales identifiziert werden. Das Protein vermittelt ein Calcium-abhängiges, nahezu vollständiges Schließen des Kanals, während Calcium-freies ApoCalmodulin keinen Effekt auf den Offenzustand hat. Mittels Fluoreszenz-Korrelations-Spektroskopie konnte gezeigt werden, dass ein IQ-Motiv als putative Calmodulin-Bindestelle im cytosolischen N-Terminus der Sec61alpha-Untereinheit Ca2 -CaM mit nanomolarer Affinität bindet, eine Interaktion mit ApoCaM hingegen erst bei wesentlich höheren Konzentrationen stattfindet. Die CaM-vermittelte, negative Feedback-Regulation des Sec61-Komplexes durch Calcium legt einen CDI- (calcium-dependent inactivation) Mechanismus nahe, der die Membranbarriere des Endoplasmatischen Retikulums auch in Anwesenheit des weiten Translokonkanals aufrecht erhält.Vergleichende Experimente haben zudem ergeben, dass der Sec61-Kanal aus Saccharomyces cerevisiae im Hinblick auf die grundsätzlichen elektrophysiologischen Eigenschaften übereinstimmende Charakteristika mit dem Komplex aus Canis familiaris zeigt.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20673号 / 医博第4283号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 横出 正之, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

No description available.

No description available.

No description available.

Phosphorylase kinase (EC 2.7.1.38) is a key enzyme involved in the regulation of the glycogenolysis pathway. It catalyzes the Ca²⁺-dependent phosphorylation and activation of the enzyme glycogen phosphorylase to make the active form glycogen phosphorylase. Phosphorylase kinase is composed of 4 subunits with a stoichiometry of (αβγδ)₄. The γ subunit is the catalytic subunit. The regulatory domain (residues 277-387) of γ contains a sequence resembling the sites phosphorylated in known γ substrates with the exception that a valine₃₃₂ occurs at the analogous position of the phosphorylated serine or threonine residue. / Ph. D.

Voltage-gated L-type calcium (Ca²⁺) channels (Ca_v1.2/1.3) are essential to neuronal and cardiac physiology. They convey extracellular Ca²⁺ after membrane depolarization, a crucial event in muscle contraction, cardiac adrenergic response, neurotransmission, memory, and learning. CaV1.2/1.3 are fine-tuned by auxiliary proteins that orchestrate Ca²⁺ influx into cells, and human variants of these proteins can disrupt channel function leading to disease. The present work probes in depth molecular mechanisms of Ca_v1.2/1.3 regulation by small cytosolic proteins calmodulin (CaM) and Leucine-rich repeat containing protein 10 (Lrrc10), as well as their relevance in physiology and pathophysiology. Chapter 1 introduces basic concepts of ion channel function, classification of voltage-gated Ca²⁺ channels, molecular components of Ca_v1.2/1.3 channel complexes, and participation of Ca_v1.2/1.3 in cardiac and neuronal physiology and disease. Chapter 2 dissects the potential contribution of a selectivity-filter gate on both VDI and CDI of Ca_v1.3 through extensive biophysical characterization, revealing asymmetric participation of conformational changes in the domain IV selectivity filter. The uncovered inactivation mechanism may be of relevance for reversing the molecular phenotype observed in Timothy syndrome, an arrhythmogenic disorder that partially stems from reduced Ca_v1.2 inactivation. Chapter 3 considers Lrrc10 as a regulatory subunit of CaV channels, uncovers molecular mechanisms, including binding interfaces that support Ca_v1.2 upregulation, and evaluates the functional consequences of human variants in Lrrc10. As Lrr proteins can interact with a wide range of targets, Chapter 4 probes the promiscuity of Lrrc10 as an ion channel modulator. Using FRET analysis, I find that Lrrc10 can, in fact, associate with various ion channels. Further analysis revealed that Lrrc10 interaction with one of its potential targets, the cardiac NaV1.5 channel, alters channel function. More broadly, these studies establish a framework to systematically screen cross talk between ion channel subunits. Finally, in Chapter 5, I leverage insights obtained from in-depth characterization of Lrrc10 modulation to engineer a genetically encoded actuator that upregulates Ca_v1.2/1.3 currents in distinct physiological settings. Altogether, this work contributes to our molecular understanding of Ca_v1.2/1.3 regulation by small cytosolic proteins, and establishes new strategies to probe and manipulate a Ca²⁺ channel function that may ultimately aid in discovering potential new targets and tools for research and therapeutics.