@phdthesis{CruzGarcia2021,
  author    = {Cruz Garcia, Yiliam},
  title     = {Interactome of the β2b subunit of L-type voltage-gated calcium channels in cardiomyocytes},
  doi       = {10.25972/OPUS-20857},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208579},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {L-type voltage-gated calcium channels (LTCC) are heteromultimeric membrane proteins that allow Ca2+ entry into the cell upon plasma membrane depolarization. The β subunit of voltage-dependent calcium channels (Cavβ) binds to the α-interaction domain in the pore-forming α1 subunit and regulates the trafficking and biophysical properties of these channels. Of the four Cavβ isoforms, Cavβ2 is predominantly expressed in cardiomyocytes. This subunit associates with diverse proteins besides LTCC, but the molecular composition of the Cavβ2 nanoenvironments in cardiomyocytes is yet unresolved. Here, we used a protein-labeling technique in living cells based on an engineered ascorbate peroxidase 2 (APEX2). In this strategy, Cavβ2b was fused to APEX2 and expressed in adult rat cardiomyocytes using an adenovirus system. Nearby proteins covalently labeled with biotin-phenol were purified using streptavidin-coated beads and identified by mass spectrometry (MS). Analysis of the in situ APEX2-based biotin labeling by MS revealed 61 proteins located in the nanoenvironments of Cavβ2b, with a high specificity and consistency in all the replicates. These proteins are involved in diverse cellular functions such as cellular trafficking, sarcomere organization and excitation-contraction coupling. Among these proteins, we demonstrated an interaction between the ryanodine receptor 2 (RyR2) and Cavβ2b, probably coupling LTCC and the RyR2 into a supramolecular complex at the dyads. This interaction is mediated by the Src homology 3 (SH3) domain of Cavβ2b and is necessary for an effective pacing frequency-dependent increase in Ca2+-induced Ca2+ release in cardiomyocytes.},
  subject      = {Calciumkanal},
  language  = {en}
}
@phdthesis{Pickel2020,
  author    = {Pickel, Simone},
  title     = {Role of the β subunit of L-type calcium channels in cardiac hypertrophy},
  doi       = {10.25972/OPUS-19282},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192829},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {L-type calcium channels (LTCCs) control crucial physiological processes in cardiomyocytes such as the duration and amplitude of action potentials, excitation-contraction coupling and gene expression, by regulating the entry of Ca2+ into the cells. Cardiac LTCCs consist of one pore-forming α1 subunit and the accessory subunits Cavβ, Cavα2δ and Cavγ. Of these auxiliary subunits, Cavβ is the most important regulator of the channel activity; however, it can also have LTCC-independent cellular regulatory functions. Therefore, changes in the expression of Cavβ can lead not only to a dysregulation of LTCC activity, but also to changes in other cellular functions. Cardiac hypertrophy is one of the most relevant risk factors for congestive heart failure and depends on the activation of calcium-dependent prohypertrophic signaling pathways. However, the role of LTCCs and especially Cavβ in this pathology is controversial and needs to be further elucidated. Of the four Cavβ isoforms, Cavβ2 is the predominant one in cardiomyocytes. Moreover, there are five different splice variants of Cavβ2 (Cavβ2a-e), differing only in the N-terminal region. We reported that Cavβ2b is the predominant variant expressed in the heart. We also revealed that a pool of Cavβ2 is targeted to the nucleus in cardiomyocytes. The expression of the nuclear Cavβ2 decreases during in vitro and in vivo induction of cardiomyocyte hypertrophy and overexpression of a nucleus-targeted Cavβ2 completely abolishes the in vitro induced hypertrophy. Additionally, we demonstrated by shRNA-mediated protein knockdown that downregulation of Cavβ2 enhances the hypertrophy induced by the α1-adrenergic agonist phenylephrine (PE) without involvement of LTCC activity. These results suggest that Cavβ2 can regulate cardiac hypertrophy through LTCC-independent pathways. To further validate the role of the nuclear Cavβ2, we performed quantitative proteome analyses of Cavβ2-deficient neonatal rat cardiomyocytes (NRCs). The results show that downregulation of Cavβ2 influences the expression of various proteins, including a decrease of calpastatin, an inhibitor of the calcium-dependent cysteine protease calpain. Moreover, downregulation of Cavβ2 during cardiomyocyte hypertrophy drastically increases calpain activity as compared to controls after treatment with PE. Finally, the inhibition of calpain by calpeptin abolishes the increase in PE-induced hypertrophy in Cavβ2-deficient cells. These results suggest that nuclear Cavβ2 has Ca2+- and LTCC-independent functions during the development of hypertrophy. Overall, our results indicate a new role for Cavβ2 in antihypertrophic signaling in cardiac hypertrophy.},
  subject      = {Herzhypertrophie},
  language  = {en}
}
@phdthesis{Chen2014,
  author    = {Chen, Wenchun},
  title     = {Studies on the role of calcium channels and the kinase domain of transient receptor potential melastatin-like 7 (TRPM7) in platelet function},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-103719},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Platelet activation and aggregation are essential processes for the sealing of injured vessel walls and preventing blood loss. Under pathological conditions, however, platelet aggregation can lead to uncontrolled thrombus formation, resulting in irreversible vessel occlusion. Therefore, precise regulation of platelet activation is required to ensure efficient platelet plug formation and wound sealing but also to prevent uncontrolled thrombus formation. Rapid elevations in the intracellular levels of cations are a core signaling event during platelet activation. In this thesis, the roles of Ca2+ and Mg2+ channels in the regulation of platelet function were investigated. Orai1, the major store-operated calcium (SOC) channel in platelets, is not only vital for diverse signaling pathways, but may also regulate receptor-operated calcium entry (ROCE). The coupling between the Orai1 signalosome and canonical transient receptor potential channel (TRPC) isoforms has been suggested as an essential step in the activation of store-operated calcium entry (SOCE) and ROCE in human platelets. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms still remains to be answered. In the first part of this thesis, the functional crosstalk between Orai1 and TRPC6 was addressed. Orai1-mediated SOCE was found to enhance the activity of phospholipases (PL) C and D, to increase diacylglycerol (DAG) production and finally to regulate TRPC6-mediated ROCE via DAG, indicating that the regulation of TRPC6 channel activity seems to be independent of the physical interaction with Orai1. Furthermore, Orai1 and TRPC6 double deficiency led to a reduced Ca2+ store content and basal cytoplasmic Ca2+ concentrations, but surprisingly also enhanced ATP secretion, which may enhance Ca2+ influx via P2X1 and compensate for the severe Ca2+ deficits seen in double mutant platelets. In addition, Orai1 and TRPC6 were not essential for G protein-coupled receptor (GPCR)-mediated platelet activation, aggregation and thrombus formation. Transient receptor potential melastatin-like 7 (TRPM7) contains a cytosolic serine/threonine protein kinase. To date, a few in vitro substrates of the TRPM7 kinase have been identified, however, the physiological role of the kinase remains unknown. In the second part of this thesis, mice with a point mutation which blocks the catalytic activity of the TRPM7 kinase (Trpm7KI) were used to study the role of the TRPM7 kinase in platelet function. In Trpm7KI platelets phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism and Ca2+ mobilization were severely impaired upon glycoprotein (GP) VI activation, indicating that the TRPM7 kinase regulates PLC function. This signaling defect in Trpm7KI platelets resulted in impaired aggregate formation under flow and protected animals from arterial thrombosis and ischemic brain infarction. Altogether, these results highlight the kinase domain of TRPM7 as a pivotal signaling moiety implicated in the pathogenesis of thrombosis and cerebrovascular events.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Fetting2011,
  author    = {Fetting, Doreen [verh: Korb]},
  title     = {Novel Cav1.2 and PMCA4b interacting PDZ domain containing proteins},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-66440},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {The voltage -gated calcium channel, Cav1.2, and the plasma membrane calcium ATPase, PMCA4b, play important roles in excitable and non-excitable cells. The central function of Cav1.2 is to regulate the calcium entry into cells upon depolarization, while PMCA4b is responsible for calcium extrusion and has an influence on cellular calcium homeostasis. Both proteins control fundamental functions in the heart and brain, but the specific functions and the precise mechanisms are still investigated. In order to identify new interaction partners that may regulate the activities of the Cav1.2 and the PMCA4b, we used three independent assays and co-localization studies. The assays, which were used are PDZ domain arrays (testing 124 different PDZ domains), GST pull-downs, and conventional immunoprecipitation assays. In the PDZ arrays, strongest interactions with Cav1.2 and PMCA4b were found for the PDZ domains of MAST-205, MAGI-1, MAGI-2, MAGI-3, and ZO-1. Additionally, we established interactions between Cav1.2 and the PDZ domains of NHERF1/2, Mint-2, and CASK. PMCA4b was observed to interact with Mint-2, and its interactions with Chapsyn-110 and CASK were confirmed. Furthermore, we validated interaction of Cav1.2 and PMCA4b with NHERF1, CASK, MAST-205 and MAGI-3 via immunoprecipitation. We also demonstrated direct interaction of the C-terminus of Cav1.2 and the PDZ domain of nNOS. We assumed that nNOS overexpression would reduce Ca2+ influx through Cav1.2. To address this question, we measured Ca2+ currents in stably transfected HEK 293 cells expressing the Cav1.2 (α1b and β2a subunit of the smooth muscle L-type calcium channel) and nNOS. It has been shown that NO modulates ion channel activity by nitrosylation of sulfhydryl groups on the channel protein. So we propose that the interaction between the C-terminus of Cav1.2 and the PDZ domain of nNOS inhibits the currents by an S-nitrosylation of the channel protein. All these interactions connect both proteins to signaling networks involved in signal transmission, cell adhesion, and apoptosis, which may help provide new hints about the physiological functions of Cav1.2 and PMCA4b in intra- and intercellular signaling.},
  subject      = {Calciumkanal},
  language  = {en}
}