Attempts were made to determine the pore properties, such as permeability, rectification and voltage-dependent block, of the putative TRPV4-TRPC1 channel. We demonstrated that this putative TRPV4-TRPC1 heterotetrameric channels displays distinct property different (although not drastically different) from TRPV4 homotetrameric channel with regard to I-V relation, kinetics of cation current, cations permeability and rectification properties. Together, the data from FRET and functional studies both suggest that heterologous expression of TRPV4 and TRPC1 can produce functional TRPV4-TRPC1 heterotetrameric channel. / Hemodynamic blood flow is one of most important physiological factors that control vascular tone. Flow shear stress acts on the endothelium to stimulate the release of vasodilators such as nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factors, causing endothelium-dependent vascular relaxation. In many cases, a key early signal in this flow-induced vascular dilation is Ca2+ influx in endothelial cells in response to flow. There is intense interest in searching for the molecular identity of the channels that mediate flow-induced Ca2+ influx. The present study aimed at identifying an interaction of TRPV4 with TRPC1, and investigating functional role of such a complex in flow-induced Ca2+ influx / In functional study, flow elicited a [Ca2+]i rise in TRPV4-expressing HEK cells. Co-expression of TRPC1 with TRPV4 markedly prolonged this [Ca2+]i transient, and it also enabled this [Ca2+]i transient to be negatively modulated by protein kinase G (PKG). Furthermore, this [Ca2+]i rise was inhibited by an anti-TRPC1 blocking antibody T1E3 and a dominant negative construct TRPC1Delta567-793. Physical interaction of TRPV4 with TRPC1 and functional role of such a complex were also found in the primary cultured rat mesenteric artery endothelial cells (MAECs) and human umbilical vein endothelial cells (HUVECs). A TRPC 1-specific siRNA was used to knock-down TRPC1 protein levels in HUVECs. Interestingly, this siRNA not only reduced the magnitude of flow-induced [Ca2+]i rise, but also accelerated the decay of flow-induced [Ca2+]i transient. Pressure myograph was used to investigate the functional role of such a complex in flow-induced vascular dilation. T1E3 also decreased flow-induced vascular dilation. Thogether, the data from endothelial cells are consistent with those in overexpressed HEK cells, supporting the notion that TRPC 1 interacts with TRPV4 to prolong the flow-induced[Ca2+]i transient, and that TRPV4-TRPC1 complex plays an important role in flow-induced vascular dilation. / In summary, my study demonstrated that TRPV4 is capable of assembling with TRPC1 to form a functional TRPV4-TRPC1 heteromeric channel. TRPV4-TRPC1 heteromeric channel can rapidly translocate to the plasma membrane after Ca 2+ depletion in intracellular stores. This TRPV4-TRPC1 heteromeric channel plays an important role in flow-induced endothelial Ca2+ influx and its associated vascular relaxation. / Ion channels are delivered to the plasma membrane via vesicle trafficking. Thus the vesicle trafficking is a key mechanism to control the amount of TRP channel proteins in the plasma membrane, where they perform their function. TRP channels in vivo are often composed of heteromeric subunits. However, up to the present, there is lack of knowledge on trafficking of heteromeric TRP channels via vesicular translocation. In the present study, we examined the effect of Ca2+ store depletion on the translocation of TRPV4-TRPC1 heteromeric channels to the plasma membrane. Experiments using total internal fluorescence reflection microscopy (TIRFM) and biotin surface labeling showed that depletion of intracellular Ca2+ stores triggered a rapid translocation of TRPV4-TRPC1 channel proteins into the plasma membrane. Fluorescent Ca2+ measurement and patch clamp studies demonstrated that store Ca2+ depletion augmented several TRPV4-TRPC1 complex-related functions, which include store-operated Ca2+ influx and cation current as well as 4alpha-PDD-stimulated Ca2+ influx and cation current. The translocation required stromal interacting molecule 1 (STIM1). Furthermore, TRPV4-TRPC1 complex is more favorably translocated to the plasma membrane than TRPC1 or TRPV4 homomers. Similar mechanisms were identified in native endothelial cells, where the TRPV4-TRPC I complex is a key component mediating flow-induced Ca2+ influx and subsequent vascular relaxation. / With the use of fluorescence resonance energy transfer (FRET), co-immunoprecipitation and subcellular colocalization methods, it was found that TRPC1 interacts physically with TRPV4 to form a heteromeric channel complex. In addition, our experimental results indicate that C-terminal and N-terminal domains of both channels are required for their interaction. / Ma, Xin. / Adviser: Yao Xiaodiang. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 109-121). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wong, Chun Kit. / "September 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Declaration --- p.vi / Abstract --- p.vii / 摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Embryonic Stem Cells (ESCs) / Chapter 1.1.1 --- Characteristics of ESC --- p.1 / Chapter 1.1.2 --- Therapeuticotential of ESCs --- p.2 / Chapter 1.2 --- 17β-estradiol (E2) / Chapter 1.2.1 --- Genomic Actions of E2 --- p.3 / Chapter 1.2.2 --- Non-genomic Actions of E2 --- p.5 / Chapter 1.2.3 --- hysiological Roles of E2 on Early Mammalian Development --- p.9 / Chapter 1.2.4 --- E2 and Cell Proliferation --- p.10 / Chapter 1.3 --- Ca2+ homeostasis / Chapter 1.3.1 --- Overview --- p.11 / Chapter 1.3.2 --- Ca2+ Signaling in mESCs --- p.14 / Chapter 1.4 --- Store-operated Ca2+ Entry (SOCE) / Chapter 1.4.1 --- Overview --- p.15 / Chapter 1.4.2 --- Store Depletion --- p.15 / Chapter 1.4.3 --- Activation of SOCE --- p.16 / Chapter 1.5 --- Molecular Identities of Store-operated Ca2+ Channels (SOCCs) on plasma Membrane / Chapter 1.5.1 --- TRPC Channels --- p.17 / Chapter 1.5.2 --- ORAI Channels --- p.18 / Chapter 1.5.3 --- Regulation of SOCCs at Different Levels --- p.18 / Chapter 1.5.4 --- Regulation of SOCE --- p.19 / Chapter 1.6 --- Nuclear Factor of Activated T-cells (NFAT) / Chapter 1.6.1 --- Overview --- p.20 / Chapter 1.6.2 --- Mechanisms of Action --- p.21 / Chapter 1.6.3 --- Functions --- p.22 / Chapter 1.7 --- Aims of the Study --- p.23 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- Maintenance of mESCs --- p.24 / Chapter 2.2 --- Cell proliferation Assay and Viability Test --- p.24 / Chapter 2.3 --- "RNAreparation, Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR)" --- p.25 / Chapter 2.4 --- Totalrotein Extraction --- p.27 / Chapter 2.5 --- Measurement of protein Concentration --- p.27 / Chapter 2.6 --- De-phosphorylation Assay --- p.28 / Chapter 2.7 --- Western Blot --- p.28 / Chapter 2.8 --- Ca2+ Measurement by Confocal Microscopy --- p.30 / Chapter 2.9 --- Ca2+ Measurement by Flow Cytometry --- p.31 / Chapter 2.10 --- siRNA Transfection --- p.31 / Chapter 2.11 --- DNAlasmid Transfection --- p.32 / Chapter 2.12 --- Molecular and Fluorescence Imaging --- p.33 / Chapter 2.13 --- Statistical Analysis --- p.34 / Chapter 2.14 --- Primers used in the Study (Table 1:Primers List) --- p.34 / Chapter 2.15 --- Drugs used in the Study (Table 2: Drugs List) --- p.36 / Chapter 2.16 --- Antibodies used in the Study (Table 3: Antibodies List) --- p.37 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Expression of SOCE in mESCs --- p.38 / Chapter 3.2 --- SOCC Blockers Attenuated mESCroliferation --- p.43 / Chapter 3.3 --- E2 Increased mESCroliferation --- p.48 / Chapter 3.4 --- E2 Increased Intracellular Ca2+ ([Ca2+]i) Level in mESCs --- p.48 / Chapter 3.5 --- E2 Increased the Amplitude of SOCE --- p.51 / Chapter 3.6 --- Increase in mESC proliferation and SOCE Caused by E2 Could be Reversed by SOCC Blocker --- p.51 / Chapter 3.7 --- Relative Expression of SOCC Candidates at mRNA Level Under the Treatment of E2 --- p.56 / Chapter 3.8 --- E2 Down-regulated the Expression of ORAI3 --- p.56 / Chapter 3.9 --- Knockdown of ORAI3 in mESCs --- p.61 / Chapter 3.10 --- Identification of NFATc3 Specific Bands --- p.63 / Chapter 3.11 --- E2 Increased the phosphorylation of NFATc3 --- p.67 / Chapter 3.12 --- Effects of 2-APB on NFATc3 phosphorylation Status --- p.67 / Chapter 3.13 --- Identification of NFATc4 Specific Bands ？ --- p.72 / Chapter 3.14 --- E2 Increased the Translocation of GFP-NFATc4 From the Cytoplasm to the Nucleus and This Effect Could be Reversed by 2-APB --- p.80 / Chapter 3.15 --- CsA Reversed E2-induced Increase in proliferation --- p.82 / Chapter CHAPTER FOUR: --- DISCUSSION / Chapter 4.1 --- Expression of SOCE in mESCs --- p.84 / Chapter 4.2 --- proliferation of mESCs Depends on SOCE --- p.85 / Chapter 4.3 --- E2 Acts an Extrinsic Factor for Stimulatingroliferation of mESCs Via SOCE --- p.87 / Chapter 4.4 --- roposed Mechanism to Show an Increment of SOCE Can be Due to a Down-regulation of ORAI3 --- p.89 / Chapter 4.5 --- Experiments Aiming to Knockdown ORAI3 --- p.92 / Chapter 4.6 --- roposed Mechanism to Show an Increment of SOCE by Other SOCC Candidates Rather than ORAI3 --- p.93 / Chapter 4.7 --- Activation of NFATc3 and NFATc4 by E2 in mESCs --- p.94 / Chapter 4.8 --- possible Downstream Targets of NFAT Responsible for E2-induced mESCs proliferation --- p.96 / Chapter CHAPTER FIVE: --- FUTUREERSPECTIVES --- p.98 / Chapter CHAPTER SIX: --- CONCLUSION --- p.100 / REFERENCES --- p.104

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Indiana University-Purdue University Indianapolis (IUPUI) / Synaptic transmission is coordinated by a litany of protein-protein interactions that rely on the proper localization and function of pre- and post-synaptic Ca2+ channels. The axonal guidance/specification collapsin response mediator protein-2 (CRMP-2) was identified as a potential partner of the pre-synaptic N-type voltage-gated Ca2+ channel (CaV2.2). CRMP-2 bound directly to CaV2.2 in two regions; the channel domain I-II intracellular loop and the distal C-terminus. Both proteins co-localized within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to EGFP caused a significant increase in Ca2+ channel current density whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2–overexpressing neurons. Both activity- and CRMP-2-phosphoryation altered the interaction between CaV2.2 and CRMP-2. I identified a CRMP-2-derived peptide (called CBD3) that bound CaV2.2 and effectively disrupted the interaction between CaV2.2 and CRMP-2. CBD3 peptide fused to the HIV TAT protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, and reversed neuropathic hypersensitivity produced by an antiretroviral drug. Unchecked Ca2+ influx via N-methyl-D-aspartate receptors (NMDARs) has been linked to activation of neurotoxic cascades culminating in cell death (i.e. excitotoxicity). CRMP-2 was suggested to affect NMDAR trafficking and possibly involved in neuronal survival following excitotoxicity. Based upon these studies, I hypothesized that a peptide from CRMP2 could preserve neurons in the face of excitotoxic challenges. Lentiviral–mediated CRMP2 knockdown or treatment with TAT-CBD3 blocked neuronal death following glutamate exposure likely via blunting toxicity from NMDAR-mediated delayed calcium deregulation. TAT-CBD3 induced internalization of the NMDAR subunit NR2B in dendritic spines without altering somal surface expression. TAT-CBD3 reduced NMDA-mediated Ca2+-influx and currents in cultured neurons. The presented work validates CRMP-2 as a novel modulator of pre- and post-synaptic Ca2+ channels and provides evidence that the TAT-CBD3 peptide could be useful as a potential therapeutic for both chronic neuropathic pain and excitotoxicity following stroke or other neuronal insults.

The role of T-type calcium channels (TTCCs) in the heart is unclear. TTCCs are transiently expressed throughout the neonatal heart during a period of rapid cardiac development. A few weeks postnatally, TTCCs are no longer found in ventricular myocytes (VMs) and calcium influx via TTCCs (ICa,T) is only detected in the SA node and Purkinje system. However, pathologic cardiac stress is associated with re-expression of TTCCs in VMs. Whether ICa,T in this setting promotes cardiac growth or exacerbates cardiac function is a topic of debate. The focus of this thesis work was to examine the effect of TTCC re-expression in the normal and diseased myocardium. Our experiments were performed in a transgenic mouse model with inducible, cardiac-specific expression of α1G TTCCs. While both the α1G and α1H TTCC subtypes re-appear during cardiac disease, we specifically evaluated the effects of α1G TTCCs since mRNA levels of this TTCC subtype are markedly elevated during cardiac pathology. We found that transgenic mice with α1G overexpression had robust ICa,T with biophysical properties similar to those published in previous studies. α1G mice had a small increase in cardiac function and showed no evidence of cardiac histopathology or increased mortality. These findings were in contrast to the phenotype of transgenic mice with augmented L-type calcium channel (LTCC) activity secondary to overexpression of the β2a regulatory subunit. While the magnitude of calcium influx in α1G and β2a VMs was similar, we found that cardiac contractility of β2a mice was significantly greater than α1G mice. Also, β2a mice had significant cardiac fibrosis, myocyte death, and premature lethality compared to the benign phenotype of α1G mice. We showed that the phenotypic differences are likely related to the differential spatial localization of T- and LTCCs. Whereas α1G TTCCs were principally localized to the surface sarcolemma, LTCCs were primarily found in the transverse tubules in close proximity to the sites of sarcoplasmic reticulum calcium release. We evaluated the effect of TTCC expression during cardiac disease by inducing myocardial infarction (MI) in α1G mice. Acutely (1-week post MI), α1G mice showed similar worsening of cardiac function and mortality rates compared to control post-infarct mice. However, α1G hearts had smaller infarct sizes which correlated with increased Akt and NFAT activation in α1G than control hearts. After chronic heart failure, i.e. 7- weeks post-infarction, α1G hearts had significant hypertrophic response as determined by increased HW/BW ratio, myocyte cross-sectional area, as well as NFAT and Akt activity. Finally, α1G mice had a small survival benefit than control mice, which while statistically non-significant, suggests that TTCC re-expression does not exacerbate cardiac function as hypothesized by some investigators. We conclude that TTCCs play a minimal role in cardiac function and activate pro-survival signaling pathways in the myocardium. / Physiology

Previous investigations on pulmonary artery smooth muscle cells have shown that nicotinic acid adenine dinucleotide diphosphate (NAADP) evokes highly localised intracellular Ca²⁺ bursts by mobilising thapsigargin-insensitive Ca²⁺ stores. Such localised Ca²⁺ signals may initiate global Ca²⁺ waves and contraction of the myocytes through the recruitment of ryanodine receptors (RyR) located on the sarcoplasmic reticulum (SR) via Ca²⁺-induced Ca²⁺-release (CICR). In this thesis I have shown that NAADP evokes localised Ca²⁺ signals through the mobilisation of a bafilomycin A1-sensitive, lysosome-related Ca²⁺ store. Lysosomal Ca²⁺ stores facilitate this process by colocalising with a subpopulation of RyRs on the surface of the SR to comprise a highly specialised trigger zone for Ca²⁺ signalling by NAADP. I have also shown that the proposed trigger zone for NAADP-dependent Ca²⁺ signalling may be formed between lysosomes and clusters of RyR subtype 3 (RyR3) located in close proximity to one another in the perinuclear region of cells. Localised Ca²⁺ bursts generated by NAADP-dependent Ca²⁺ release from acidic Ca²⁺ stores and subsequent CICR via RyR3 on the SR may then amplify Ca²⁺ bursts into a propagating Ca²⁺ signal by recruiting clusters of RyR subtype 2 (RyR2) located in the perinuclear and extra-perinuclear regions of the cell. The presence of this trigger zone may explain, in part, why Ca²⁺ bursts by NAADP induce, in an all-or-none manner, global Ca²⁺ signals by CICR via RyRs on the SR. Consistent with a role for NAADP and lysosomes as a discrete and agonist-specific Ca²⁺ signalling pathway utilised by vasoconstrictors, I have shown that endothelin-1 (ET-1), but not phenylephrine or prostaglandin-F2α, mobilises Ca²⁺ stores by NAADP, and that ET-1 initiates Ca²⁺ signalling by NAADP in a receptor subtype-specific manner through the activation of ETB receptors. These findings further advance our understanding of how that spatial organisation of discrete, organellar Ca²⁺ stores underpin the generation of differential Ca²⁺ signalling patterns by different Ca²⁺-mobilising messengers.

published_or_final_version / abstract / Physiology / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Dans la fibre musculaire squelettique, le calcium activateur de la contraction musculaire provient du réticulum sarcoplasmique (RS). Parallèlement, un influx calcique est connu pour se développer à travers la membrane plasmique au repos et en activité, mais son rôle physiologique est méconnu. Le travail présenté vise par des approches électrophysiologiques, cellulaires et moléculaires à caractériser les différentes voies d’influx de calcium et leurs mécanismes de régulation dans la fibre musculaire squelettique de souris adulte normale et pathologique. En combinant la technique de mesure d’extinction de fluorescence du Fura-2 par le manganèse à la technique de potentiel imposé, nos résultats démontrent dans une première partie que le calcium rentre au repos de manière passive selon son gradient électrochimique sans générer de courant détectable, tandis qu’en activité un influx activé par la déplétion du RS et un influx activé par la dépolarisation, tous deux électriquement silencieux, se développent en parallèle de l’entrée médiée par les canaux calciques voltage- dépendants de type L. La deuxième partie du travail s’intéresse à la protéine TRPC1 dont le rôle fonctionnel reste controversé dans la fibre musculaire. Contrairement aux résultats décrits dans la littérature, nos expériences de surexpression indiquent que TRPC1 s’exprime au niveau du RS longitudinal où il génère une fuite de calcium. Dans une dernière partie, il est décrit que l’amplitude des courants calciques voltage-dépendants de type L est réduite dans les fibres musculaires d’un modèle de souris présentant une myopathie centronucléaire liée à un déficit en une phosphatase lipidique, la myotubularine. / In skeletal muscle, calcium in charge of activation of the contraction is released from the sarcoplasmic reticulum (SR). In parallel, a calcium influx is known to occur through the plasma membrane at rest and during activity, but its role remains elusive. The present work aims at characterizing the different calcium influx pathways and the mechanisms that regulate their activity in normal and pathological adult mouse skeletal muscle fiber using electrophysiological, cellular and molecular approaches. By combining the technique of manganese quenching of Fura-2 fluorescence and voltage clamp, our data first demonstrate that calcium enters muscle cell in a passive manner driven by the electro-chemical gradient without generating current, while during activity, a calcium influx activated by SR depletion and an influx evoked by depolarization, both electrically silent, occur in parallel with the calcium entry supported by voltage-dependent L-type calcium channels. The second part of the work investigates the properties of the TRPC1 protein whose functional role remains controversial in skeletal muscle. In contrast to data reported in the literature, our TRPC1 over- expression experiments indicate that TRPC1 is localized in the longitudinal SR where it operates as a calcium leak channel. In the last part of the work, we describe that the amplitude of the voltage-dependent L-type calcium channels is reduced in the muscle fiber from a murine model of centronuclear myopathy induced by a deficiency in the lipid phosphatasemyotubularin.

Cardiovascular disease (CVD) claims more lives than any other disease in the world. Although numerous biological pathways share the blame, ventricular tachyarrhythmia (VT) is estimated to account for ~25% of all CVD deaths. A complete understanding of the molecular mechanisms underlying VT is unknown but recent studies have linked VT to improper calcium handling in the heart (canine). The principle calcium regulator in the muscle cell is the calcium ion release channel (aka RyR). Numerous endogenous and exogenous compounds can affect the way the RyR regulates calcium. In particular, abnormal levels of oxidants (reactive oxygen species) can oxidize critical thiol groups on the RyR and modulate its activity. Interestingly, high levels of oxidants are also associated with numerous bodily disease states including cancers, muscle fatigue/failure, and CVD. In this thesis, two important dietary antioxidant compounds, the anthocyanidins pelargonidin and delphinidin, are evaluated for their effects on regulating the transport of calcium through the calcium release channel (RyR1) of the sarcoplasmic reticulum of skeletal muscle. Pelargonidin and delphinidin are structurally similar with delphinidin only differing from pelargonidin by the addition of two hydroxyl groups. Both compounds undergo time dependent structural changes in aqueous solutions at physiological pH and a mixture of more than four structures of each compound can be present in solution simultaneously. Pelargonidin and delphinidin show distinct differences in their calcium flux regulating effect on the RyR1. Delphinidin stimulates calcium flux and RyR1 activity where as pelargonidin can cause both inhibition and stimulation of the RyR1. The strength of stimulation and inhibition of calcium transport through the RyR by delphinidin and pelargonidin may be attributed to the structural and chemical changes in those compounds that occur in solutions near physiological pH and the subsequent chemical characteristics of the diverse set of structures that are simultaneously present in solution.