Ionized calcium (Ca2+) is a signaling messenger that controls numerous cellular processes essential for life. The fidelity of Ca2+ signaling depends on the mechanisms that dynamically regulate its cytosolic concentration and maintain it at a low level in a resting cell. Plasma Membrane Ca2+ ATPase (PMCA) is a high affinity Ca2+ extrusion pathway involved in Ca2+ homeostasis and signal transduction. PMCA are encoded by 4 genes (PMCA1-4), which are expressed in a tissue dependent manner. The diversity of PMCA isoforms is further increased by alternative splicing. Changes in PMCA activity occur in heart failure and hypertension. Specific inhibitors of other ion transporters such as thapsigargin and digoxin, have made their mark in cell biology, but the currently used inhibitors of PMCA (vanadate and eosin) are non-specific. Thus, selective inhibitors of PMCA are needed to discern its role in Ca2+ signaling in physiology and pathophysiology. We introduced the concept of caloxins - peptides that specifically inhibit the activity of PMCA by binding to one of its five extracellular domains (exdoms) 1 to 5. The earlier caloxins including 2a1 and 3a1 were obtained by screening a phage display random 12-amino acid peptide (Ph.D-12) library for binding to synthetic peptides based on the exdom sequences. However, they all had low affinity. The objective of this research was to develop caloxins with high affinity and PMCA4 isoform selectivity. A two-step screening method was developed to screen the Ph.D-12 library to first bind to the synthetic exdom of PMCA4, followed by affinity chromatography using PMCA protein purified from human erythrocyte ghosts (mainly PMCA4). This method was used to obtain caloxins 1b1 and 1b2 to bind to the N and C-terminal halves of the exdom 1 of PMCA4, respectively. Both caloxins 1b1 and 1b2 had a 10-fold higher affinity than the prototype caloxin 2a1 and showed slight PMCA4 isoform preference. To engineer inhibitors with greater affinity and PMCA4 isoform selectivity, Ph.D caloxin 1b1 like peptide library was constructed. Most of the peptides expressed in this library differed from caloxin 1b1 in 0, 1, 2 or 3 amino acid residues at random. The library was screened to obtain several peptides one of which was caloxin 1c2. Caloxin 1c2 had 200-fold higher affinity than caloxin 2a1 and was isoform selective, with greater than 10-fold affinity for PMCA4 than for PMCA isoforms 1, 2 or 3. Thus, caloxin 1c2 is the first high affinity PMCA inhibitor that also is selective for an individual PMCA isoform. The second aim of this research was to establish that caloxin 1c2 binds to PMCA protein in erythrocyte ghosts. Two photoreactive caloxin 1c2-derivatives containing the photoactivable residue benzoylphenylalanine (Bpa) and a C-terminal biotin tag were used. Bpa substituted tryptophan at position 3 (3Bpa1c2-biotin) and serine at position 16 (16Bpa1c2-biotin) in caloxin 1c2. Both the derivatives inhibited PMCA activity in the erythrocyte ghosts. The intensity of the biotin label in the photolabeled erythrocyte ghosts was much stronger with 3Bpa1c2-biotin, which was then used in the subsequent experiments. The photolabeled proteins in erythrocyte ghosts were detected as a 250-270 kDa doublet in Western blots using streptavidin and the PMCA specific antibody. The degree of photolabeling depended on the UV-crosslinking time, and on the concentrations of 3Bpa1c2-biotin and the ghost protein. The selectivity of the photolabeling site was confirmed by decreased photolabel incorporation at 250-270 kDa doublet in the presence of excess caloxin 1c2 and the synthetic exdom 1X peptide of PMCA4. The photolabeled erythrocyte ghosts were solubilized and analyzed by immunoprecipitation with the PMCA specific antibody. The immunoprecipitate showed a 250-270 kDa doublet in Western blots using streptavidin. This confirmed that PMCA protein was photolabeled by the photoreactive derivatives of caloxin 1c2. Thus, caloxin 1c2 inhibits PMCA activity by binding to the exdom 1X of PMCA4. My work in M.Sc. initiated the concept of caloxins in the literature. This research has taken it to the stage where we can obtain caloxins selective for individual PMCA isoforms. This contrasts with the relative paucity of inhibitors specific for individual isoforms of other ion pumps. The high affinity isoform selective caloxin 1c2 and previous caloxins are being used to study PMCA physiology in our lab and by other researchers. Since caloxins act when added extracellularly and it is possible to obtain PMCA isoform selective caloxins, it is anticipated that they will aid in understanding the role of PMCA in signal transduction and homeostasis in health and disease. / Thesis / Doctor of Philosophy (PhD)

The kisspeptin receptor (KISS1R), functioning as a metastasis suppressor and gatekeeper of GnRH neurons, is a potent activator of intracellular Ca2+. The surge in cytoplasmic Ca2+ mediates the exocytosis of GnRH from GnRH neurons. However, the regulatory processes which enable KISS1R to sense increasing intracellular Ca2+ and avoid Ca2+ excitotoxicity via a signalling off-switch mechanism remain unclear. This thesis provides evidence for the interaction between KISS1R and the Ca2+ regulated proteins of calmodulin (CaM), and αCa2+/CaM-dependent-protein kinase II (α-CaMKII). Binding of CaM to KISS1R was shown with three independent approaches. Firstly, cell-free spectrofluorimeter assays showed that CaM selectively binds to intracellular loop (IL) 2 and IL3 of the KISS1R. Secondly, KISS1R co-immunoprecipitation experiments identified ligand/Ca2+-dependent binding of KISS1R to HEK-293 endogenous CaM. Thirdly, confocal experiments showed CFPCaM co-localises with YFP-KISS1R. The functional relevance of CaM binding was examined with alanine substitution of critical residues of the CaM binding motifs in IL2 and IL3 of KISS1R. This approach revealed that the receptor activity (relative maximum responsiveness) was increased in the mutated residues of the juxtamembrane regions of IL3 and the N-terminus of IL2 relative to wild-type KISS1R. The Ca2+/CaM regulated αCaMKII was also found to interact with KISS1R by selectively phosphorylating T77 of IL1. Phosphomimetic mutations of T77 into E or D created a receptor that was unable to elicit inositol phosphate production upon ligand stimulation. Finally, in vivo studies using ovariectomised rats that were intracerebroventricularly administered with a cell-permeable αCaMKII inhibitor augmented the effects of kisspeptin ligand stimulation of plasma luteinizing hormone levels. Taken together, this thesis demonstrates that the KISS1R-G protein coupling is regulated by Ca2+-dependent CaM binding and αCaMKII-mediated KISS1R phosphorylation.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal. / Dans une situation d'apprentissage, il a été proposé que les neurones recrutés et les synapses activées pour accomplir cet exercice mental se modifient afin de laisser, à l'échelle cellulaire, une trace mnésique. Les récents et nombreux travaux de recherche en neurobiologie de la mémoire ont permis de confirmer l'existence potentielle d'une partie de ces processus cognitifs hypothétiques. Entre autres, il a été démontré que les changements neuronaux survenant lors de divers phénomènes de modulation de la fonction synaptique (plasticité synaptique) seraient, en partie, le résultat d'une modification des propriétés des récepteurs glutamatergiques de type AMPA (a-amino-3-hydroxy-5-méthyl-4-isoxazolepropionate) par des processus enzymatiques dépendants de l'ion calcium (Ca2+). Cependant, la caractérisation des effets de plusieurs conditions (pathologies, comparaisons interespèces, modifications génétiques) sur les capacités mnésiques des sujets expérimentaux et sur les phénomènes de plasticité synaptique observés dans leur cerveau a été et est encore principalement effectuée par l'utilisation de souris, tandis que les données sur les propriétés de liaison des récepteurs AMPA sont, dans l'ensemble, tirées d'expériences effectuées avec des rats. Par conséquent, la caractérisation des mécanismes de régulation des propriétés de liaison des récepteurs AMP A chez la souris s'avère nécessaire afin de faciliter et d'approfondir la compréhension des processus cellulaires impliqués dans l'expression des différentes formes de plasticité synaptique associées aux circuits neuronaux glutamatergiques. Durant mes travaux de maîtrise, des analyses qualitatives et quantitatives de la liaison de ligands tritiés sur des coupes de cerveaux congelés de différentes souches de souris ont été effectuées afin de déterminer comment l'exposition de ces coupes à des ions calcium (traitement calcique) est en mesure de modifier les propriétés de liaison des récepteurs glutamatergiques AMP A. Les résultats obtenus avec la souche B6C3Fl montrent que le Ca2+ a des effets sur les propriétés de liaison de ces récepteurs qui sont différents d'une région cérébrale à l'autre : la liaison est augmentée dans l'hippocampe, diminuée dans le cortex et le striatum, et inchangée dans le thalamus. De plus, ces effets semblent spécifiques aux récepteurs AMPA puisque le traitement calcique des coupes de cerveau n'a pas induit de changements de la liaison de ligands tritiés aux récepteurs NMDA (N-méthyl-D-aspartate), un autre type de récepteurs glutamatergiques. L'analyse de courbes de saturation de la liaison de l' AMPA tritié a révélé que la hausse de la liaison induite par le Ca2+ dans la région CA1 radiatum de l'hippocampe est due à un changement dans le nombre maximal de sites de liaison (Bmax), alors que la baisse de la liaison observée dans le cortex et induite par le Ca2+ semble être le résultat d'une diminution de l'affinité (exprimée par le Kt) des récepteurs AMPA envers le ligand. Cette régulation calcique à la baisse de l'affinité des récepteurs AMP A dans le cortex et le striatum semble impliquer l'activation de la phospholipase A2 et de la voie lipoxygénase du métabolisme de l'acide arachidonique puisqu'elle est bloquée par des inhibiteurs de ces systèmes enzymatiques.

According to the centers for disease control and prevention (CDC) and the world healthorganization (WHO), heart disease and immune related diseases such as diabetes and cancer areamong the leading causes of death around the world. Thus, the regulation of the function ofimmune cell plays a key role in health and disease. Calcium (Ca2+) ions play a critical role inimmune cell activation, function and in a robust immune response. Defects in Ca2+ signalinginfluences the development of cardiac disease, Alzheimer disease, immune cell metabolism,muscle dysfunction, and cancer. Each immune cell is unique in its activation and function,making it relevant to understand how activation of each type of immune cell is regulated. Herewe describe the role of the nBMP2 protein in macrophage activation and function and the role ofthe CD5 co-receptor in helper T cell activation and function.The nuclear bone morphogenetic protein 2 (nBMP2) is the nuclear variant of the bonemorphogenetic protein 2 (BMP2), a growth factor important in heart development, neurogenesis,bone, cartilage and muscle development. To better understand the function of nBMP2, transgenicnBMP2 mutant mice were generated. These mice have a slow muscle relaxation and cognitivedeficit caused in part by abnormal Ca2+ mobilization. Mutant nBMP2 mice also have an impairedsecondary immune response to systemic bacterial challenge. Here we have further characterizedmacrophage activation and function from mutant nBMP2 mice before and after bacterialinfection. We describe how nBMP2 influences the Ca2+ mobilization response and phagocytosisin macrophages, revealing a novel role of the nBMP2 protein in immune cell regulation.CD5 is a surface marker on T cells, thymocytes, and the B1 subset of B cells. CD5 isknown to play an important role during thymic development of T cells. CD5 functions as anegative regulator of T cell receptor (TCR) signaling and fine tunes the TCR signaling response.Here we describe our characterization of CD5 regulation of Ca2+ signaling in naïve helper Tcells. We also outline our findings examining how CD5-induced changes in helper T cellactivation influence other biological processes such as immune cell metabolism, the diversity ofthe gut microbiome, and cognitive function and behavior. Thus, this work elucidates theinfluence of the CD5 co-receptor on the functional outcomes in multiple systems when CD5 isaltered.