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Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmacology and Toxicology. Title from resource description page. Includes bibliographical references.

La contraction musculaire est provoquée par un relâchement massif de calcium à partir du reticulum sarcoplasmique (RS) des cellules musculaires. Ce relâchement de calcium réalisé par le récepteur de la ryanodine (RyR1), s'effectue dans des structures membranaires spécialisées et très organisées : les triades. Cette architecture spécifique est essentielle à l'activité correcte de RyR1. Cependant, les mécanismes moléculaires mis en jeu dans la formation et le maintien des triades ne sont pas connus. La triadine, qui est une protéine localisée dans la membrane du RS et qui est associée à RyR1, pourrait jouer un rôle dans la structure du reticulum sarcoplasmique pour permettre un relâchement de calcium efficace. L'équipe a montré que l'ablation du gène de la triadine chez la souris induisait une altération des relâchements de calcium et une modification de la forme des triades.Nous avons montré que la triadine pouvait indirectement interagir avec les microtubules et qu'elle pourrait ancrer le RS aux microtubules (Fourest-Lieuvin, J Cell Science, 2012). Par analyse en spectrométrie de masse des protéines co-immunoprécipitées avec la triadine, nous avons identifiéun nouveau partenaire de la triadine, CLIMP-63 qui pourrait être impliqué dans cette fonction. CLIMP-63 est décrite comme une protéine capable d'ancrer le reticulum aux microtubules et de maintenir la forme du reticulum endoplasmique. Nous avons ensuite confirmé son interaction avec la triadine par différentes approches dans différents modèles cellulaires. L'étude et la caractérisation de CLIMP-63 dans le muscle sont tout à fait innovantes et nous avons étudié les conséquences de l'association triadine/CLIMP-63 pour la fonction du muscle et dans la formation ou la maintenance des triades. / Muscle contraction is achieved when an efficient excitation signal at the plasma membrane triggers intracellular calcium release. This process called “excitation-contraction (E-C) coupling” relies on a macromolecular protein complex, spanning the plasma membrane and the sarcoplasmic reticulum (SR), containing the calcium channel of the SR, the ryanodine receptor (RyR1). This calcium release complex is present exclusively in highly organized membrane structures called triads. A triad is composed of two SR terminal cisternae surrounding a plasma membrane transverse-tubule.This architecture is essential to sustain the activity of the calcium channel RyR1, which is located in the membrane of SR terminal cisternae. However, little is known about the molecular mechanisms allowing the formation and maintenance of SR terminal cisternae. Triadin is a member of this complex, present in the SR membrane and interacting with RyR1. Deletion of the triadin gene leads to partial disorganisation of SR membranes in skeletal muscles, with abnormal orientation of part of the triads. Triadin could play a role in the structure of sarcoplasmic reticulum to allow efficient E-C coupling. We have shown that triadin could indirectly interact with the microtubules, and therefore anchor the sarcoplasmic reticulum to the microtubule network (Fourest-Lieuvin, J Cell Science, 2012). Using mass spectrometry analysis of proteins co-immunoprecipitated with triadin, we have identified a new partner of triadin, CLIMP-63 which could be involved in this function. CLIMP-63 is a shaping protein able to mediate the anchoring of the reticulum to microtubules and to maintain the shape of endoplasmic reticulum. We have dissected the interacting domains between CLIMP-63 and triadin, and study the consequences of this association for muscle function, and triad formation or maintenance.

The sarcoplasmic reticulum is an internal membrane system that controls the Ca²⁺ concentration inside muscle cells, and hence the contractile state of both skeletal and cardiac muscle. A key protein that that regulates the Ca²⁺ concentration in this membrane is known as the calcium release channel (CRC). The effects on Ca²⁺ dependent activation is of major importance in the study of CRC since other channel modifiers cannot effect the channel in the absence of Ca²⁺, or they require Ca²⁺ for maximum results. In this study of the high-affinity Ca²⁺ binding site, expected increases in total binding and shifts in the sensitivity of the channel to Ca²⁺ were observed when the pH increased or the solution redox status became more oxidative. Ranolazine, a drug used for treating Angina Pectoris (chest pain), desensitized the cardiac CRC activation but had no effect on the skeletal CRC. This selective desensitization may be the cause of Ranolazine's beneficial therapeutic effects. Both Ranolazine, and homocystein thiolactone (HCTL), a naturally occurring derivative of homocysteine, alters Ca²⁺ dependent activation by calcium without changing the number of channels found in the open state. Surprisingly the effect of HCTL was observed only in a reduced redox potential which leads to speculation that the formation of an alpha-carbon radical by HCTL on the cardiac CRC only occurs if select thiols are in a reduced state.

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Well-regulated Ca2+ signalling is essential for every living organism, and disruption of this signalling can lead to diseases including heart failure, neurological disorders and diabetes. Intracellular Ca2+ levels are regulated by influx of extracellular Ca2+ through channels located in the cell membrane. In addition, release of Ca2+ from intracellular stores also plays an important role in controlling intracellular Ca2+ concentration. Of the three types of intracellular Ca2+ stores that have been characterised those with D-myo-Inositol 1,4,5 trisphosphate receptors (InsP3Rs) showed a close relationship with cell proliferation. Hence, selective blockage of InsP3Rs will allow better understanding of Ca2+ signalling and might also unveil novel treatment for cancers, in the long term. There were no selective InsP3Rs antagonists known at the start of these studies. Based on the crystal structure of InsP3Rs bound to InsP3 and SAR studies of InsP3, we designed and tested several InsP3 analogues.1 Compound 15, 16 and 23 acted as InsP3R antagonists, though their selectivity for InsP3Rs was not completely determined. Furthermore, we also attempted to improve the potency of 16 via substitution at the 1-postion phosphate. By considering the interaction formed between adenophosphostins and InsP3Rs compounds (53-55) were designed and synthesised. In addition, analogues of compound 92, selected from an in silico screen, have led to the discovery of another novel scaffold that acts as an InsP3R antagonist.

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca2+] in the vicinity of plasmalemmal Ca2+ channels due to Ca2+ influx, elicits exocytosis. This dissertation examines the effect on both spontaneous and elicited exocytosis of a rise in focal cytosolic [Ca2+] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca2+ syntillas. Ca2+ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (Scintilla, Latin for spark, found in nerve terminals, normally synaptic structures.) We have also observed Ca2+ syntillas in mouse adrenal chromaffin cells (ACCs). Here the effect of Ca2+syntillas on exocytosis is examined in ACCs, which are widely used as model cells for the study of neurosecretion. Elicited exocytosis employs two sources of Ca2+, one due to influx from the cell exterior through voltage-gated Ca2+ channels (VGCCs) and another due to release from intracellular stores. To eliminate complications arising from Ca2+ influx, the first part of this dissertation examines spontaneous exocytosis where influx is not activated. We report that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine after which syntillas were decreased. We conclude that Ca2+syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas. The second part of this dissertation examines the role of syntillas in elicited exocytosis whereby Ca2+ influx is activated by physiologically relevant levels of stimulation. Catecholamine and neuropeptide release from ACCs into the circulation is controlled by the sympathetic division of the Autonomic Nervous System. To ensure proper homeostasis tightly controlled exocytic mechanisms must exist both in resting conditions, where minimal output is desirable and under stress, where maximal, but not total release is necessary. It is thought that sympathetic discharge accomplishes this task by regulating the frequency of Ca2+ influx through VGCCs, which serves as a direct trigger for exocytosis. But our studies on spontaneous release in ACCs revealed the presence of Ca2+ syntillas, which had the opposite effect of inhibiting release. Therefore, assuming Ca2+-induced Ca2+ release (CICR) via RYRs due to Ca2+ influx through VGCCs, we are confronted with a contradiction. Sympathetic discharge should increase syntilla frequency and that in turn should decreaseexocytosis, a paradox. A simple “explanation” might be that the increase in syntillas would act as a brake to prevent an overly great exocytic release. But upon investigation of this question a different finding emerged. We examined the role of syntillas under varying levels of physiologic stimulation in ACCs using simulated action potentials (sAPs) designed to mimic native input at frequencies associated with stress, 15 Hz, and the basal sympathetic tone, 0.5 Hz. Surprisingly, we found that sAPs delivered at 15 Hz or 0.5 Hz were able to completely abolish Ca2+ syntillas within a time frame of two minutes. This was not expected. Further, a single sAP is all that was necessary to initiate suppression of syntillas. Syntillas remained inhibited after 0.5 Hz stimulation but were only temporarily suppressed (for 2 minutes) by 15 Hz stimulation, where global [Ca2+]i was raised to 1 – 2 μM. Thus we propose that CICR, if present in these cells, is overridden by other processes. Hence it appears that inhibition of syntillas by action potentials in ACCs is due to a new process which is the opposite of CICR. This process needs to be investigated, and that will be one of the very next steps in the future. Finally we conclude that syntilla suppression by action potentials is part of the mechanism for elicited exocytosis, resolving the paradox. In the last chapter speculation is discussed into the mechanisms by which physiologic input in the form of an action potential can inhibit Ca2+ syntillas and furthermore, how the Ca2+ syntilla can inhibit exocytic output.

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