Invertebrate T-type calcium channels cloned from the great pond snail, Lymnaea Stagnalis (LCav3) possess highly sodium permeant ion channel currents by means of alternative splicing of exon 12. Exon 12 is located on the extracellular turret and the descending helix between segments 5 and segments 6, upstream of the ion selectivity filter in Domain II. Highly-sodium permeant T-type channels are generated without altering the selectivity filter locus, the primary regulatory domain known to govern ion selectivity for calcium and sodium channels. Comparisons of exon 12 sequences between invertebrates and vertebrate T-type channels reveals a conserved pattern of cysteine residues. Calcium-selective mammalian T-type channels possess a single cysteine in exon 12 in comparison to invertebrate T-type channels with either a tri- or penta- cysteine framework. Cysteine residues in exon 12 were substituted with a neutral amino acid, alanine in LCav3 channels harbouring exon 12a and 12b to mimic the turret structure of vertebrate T-type channels. The results generated T-type channels that were even more sodium-permeable than the native T-type channels in snails. Furthermore, permeant divalent ions similar in structure to calcium (eg. barium) were unable to sufficiently block the monovalent ion current of channels lacking cysteines in Domain II, suggesting that the pore is highly sodium permeant, and has weak affinity and block by permeant divalent ions other than calcium. Besides ion selectivity, the cysteine mutated T-type channels were 10 to 100 fold more sensitive to inhibition by nickel and zinc, respectively. The cysteine mutation data highly suggests that the cysteines form an extracellular structure that regulates ion selectivity and shields T-type channels from block by nickel and zinc. In addition, we replaced exon 12 from the sodium permeant snail T-type channel with exon 12 from human Cav3.2 channels. The snail T-type channel with exon 12 from human T-type channels produced a T-type channel that was modestly sodium permeable, but did not confer the high calcium permeability of Cav3.2 channels. These findings suggest that the cysteine containing extracellular domains in exon 12 are not sufficient to generate calcium selective channels similar to human Cav3.2 and likely work in concert with other extracellular domains to regulate the calcium or sodium selectivity of T-type channels.

Cardiovascular disease remains the number one cause or mortally in the western world. Heart failure is the most rapidly growing cardiovascular disease (Hobbs, 2004; Levy, et al., 2002). Heart failure, by definition, is progressive deteriorating function of the heart due to progressive cardiac myocytes loss. Though after decades of endeavor of searching the pathophysiology and treatments for heart failure, it remains highly lethal. Therefore, it is vital to find novel therapies to help treat such chronic disease. Replace the lost cardiomyocyte with new ones could restore cardiac function and reduce mortality. The purpose of this study is to investigate on how TTCCs (T-type calcium channels) affect cardiomyocyte proliferation. In mice after birth, the major TTCC expressed in the heart is Cav3.1/α1G, and therefore we used Cav3.1/α1G transgenic (TG), knockout (-/-) and wild type mice respectively to define the role of TTCC in cardiomyocyte proliferation. In neonatal mouse ventricular myocyte (NMVMs) right after birth, there is almost no TTCC after birth in α1G-/- NMVMs, whereas there are around 35% NMVMs in wild type (WT) show TTCC. On day 7 after birth, there are no T-type calcium currents in both α1G-/- NMVMs and WT NMVMs. Using BrdU, a DNA synthesis marker, we identified plenty of BrdU positive cardiomyocyte during the first seven days after birth. Cardiomyocyte is special due to its double nucleation property. Our cell cycle studies showed that there is significant difference in cell cycle distribution between α1G-/- and WT NMVMs on day seven after birth. Significantly more NMVMs are arrested in G1 phase in α1G-/-, compared to WT NMVMs. Even until 2 month old, there are still significantly more mono-nucleated cardiomyocyte in α1G-/- than in WT. In conclusion, all these evidence showed that blocking T-type calcium channel could partially prevent binucleation from happening and stop cardiomyocytes withdrawal from cell cycle. Mononucleated cardiomyocyte is still able to proliferate. Hence, mononucleated cardiomyocytes in adult still have potential to proliferation because these cardiomyoctes are arrested in their cell-cycle before their terminal differentiation, which could offer a novel approach for cardiac repair. / Physiology

No / T-type Ca2+ channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca2+ channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca2+ channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca2+ channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca2+ channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca2+ channel-mediated proliferation by H2S is independent of the channels’ sensitivity to H2S. / This work was supported by the British Heart Foundation (PG/11/84/29146).

Le traitement et la gestion de la douleur sont depuis toujours une priorité pour le corps médical. Malgré leur importance pour la qualité de vie, les analgésiques couramment utilisés possèdent un ratio bénéfice/risque faible. La recherche de nouveaux concepts thérapeutiques pour lutter contre la douleur est donc une priorité. Afin de répondre à ce besoin, il faut d'abord comprendre les mécanismes de la perception de la douleur ainsi que, plus globalement, ceux permettant de percevoir son environnement. Dans ce contexte, de nombreuses études ont mis en évidence l'implication du canal calcique à bas seuil Cav3.2 dans les voies de la transmission de l'information douloureuse. Il représente donc une cible de choix pour le traitement de la douleur mais l'identité des neurones exprimant ces canaux ainsi que la fonction de Cav3.2 dans la physiologie des neurones sensoriels étaient jusqu'à présent inconnues. Au cours de cette thèse nous avons dans un premier temps décrit un nouvel inhibiteur des canaux calciques à bas seuil : le TTA-A2. Nous avons ainsi démontré que le TTA-A2 est un inhibiteur spécifique des canaux Cav3.1, Cav3.2, et Cav3.3. Il permet de diminuer l'excitabilité des neurones sensoriels exprimant Cav3.2, ce qui provoque une analgésie sur des animaux sains et pathologiques. Dans un deuxième temps nous nous sommes servis de ce nouvel outil en parallèle d'un nouveau modèle murin possédant une étiquette fluorescente (Knock in GFP) sur le canal Cav3.2 pour explorer la localisation et la fonction de Cav3.2 dans les neurones sensoriels. Nous avons ainsi découvert que Cav3.2 est exprimé dans des mécanorécepteurs à bas seuil impliqués dans la perception des stimuli mécaniques et thermiques nocifs ou non-nocifs. Le canal en lui-même se trouve aux endroits clés de la genèse et de la propagation du message nerveux périphérique, et module le seuil et la vitesse de conduction des potentiels d'action. Replacé dans le contexte de la bibliographie, l'ensemble de nos résultats montre que Cav3.2 permet de donner la modalité à bas seuil aux neurones l'exprimant. / Pain management and treatment have always been a priority for life quality. Despite this fact, analgesics commonly used present a bad benefice/risk ratio. Discovery of new therapeutic concepts to fight pain is highly required. To complete this task, we first need to better understand pain perception mechanisms, and more globally, mechanisms involved in the perception of our environment. In this context, numerous studies have shown that low threshold calcium channels Cav3.2 are involved in pain information transmission. Thus, it represents a good target for the treatment of pain. However, neuronal identity of Cav3.2-expressing sensory neurons and Cav3.2 functions in neuronal physiology are unknown. During this PhD we first described a new low voltage activated channel antagonist named TTA-A2. We demonstrated that TTA-A2 is a powerful nanomolar specific agonist of Cav3.1, Cav3.2 and Cav3.3. This molecule is able to reduce excitability in sensory neurons expressing Cav3.2, and is able to generate a strong analgesic effect on naive and pathologic animals. In the other part of this PhD, we used this new tool combined to a new transgenic mouse that expressed Cav3.2 tagged with a fluorescent protein (Knock-in GFP). With these new tools we discovered that Cav3.2 is expressed in low threshold mechanoreceptors involved in detection of painful and non painful mechanical and thermal stimuli. Cav3.2 itself is expressed at key localisations that allow action potential generation and propagation, and modulate threshold and speed conduction of action potential. Taken together, these results show that Cav3.2 gives the “low threshold” modality to neurons.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Three research projects are described in this dissertation, and they are: (i) discovery of piperidine derivatives as T-type calcium channel inhibitors for the treatment of epilepsy and neuropathic pain and as protein disulfide isomerase inhibitors for the treatment of influenza viral infection; (ii) discovery of peptide-based calcitonin gene-related peptide receptor antagonists for the treatment of inflammatory pain; and (iii) synthesis of substituted 6-(dimethylamino)-2-phenylisoindolin-1-ones for the inhibition of luciferase. T-type calcium channels are important regulators of nervous system, and upregulated T-type calcium channel activities have been found to link to various types of neurological disorders, such as epilepsy and neuropathic pain. To discover novel T-type calcium channel blockers, a series of 1,4-disubstituted piperidine derivatives were designed and synthesized. Among them, compound 1-4 was found to be a good T-type calcium channel inhibitor with an IC₅₀ of 1 nM for Ca[subscript v]3.2 inhibition. It also showed 86% suppression of seizure induced death in mice and good in vivo analgesic effects on both thermal and mechanical pain thresholds in Spared Nerve Injury rat models. Therefore 1-4 can potentially be used as a T-type calcium channel blocker in the treatment of epilepsy and neuropathic pain. Influenza is a respiratory viral infection. Since viruses rely on host cell proteins for their entry, survival and replication, development of drugs targeting host cell proteins has identified as an effective strategy in controlling viral infections. We synthesized a series of 1,4-disubstituted piperidine derivatives for the inhibition of protein disulfide isomerase enzyme and influenza. Among them, 1-29 was found to possess strong anti-influenza activity (EC₅₀ = 2.5 µM). This suggests the potential use of piperidine scaffold in designing anti-influenza drugs in future. Calcitonin gene-related peptide (CGRP) receptor antagonism has been identified as a successful approach for the treatment of inflammatory pain. Therefore, a novel class of peptide antagonists of CGRP receptor was synthesized and screened for their binding affinities to the CGRP receptor and their analgesic effects on inflammatory-induced pain in rats. Among them, peptide 2-3 showed a higher binding affinity towards the CGRP receptor than previously reported peptide antagonists and exhibited analgesic effects up to 2 h in both Aδ and c-fiber pain tests. Therefore 2-3 indicates its potential use as a CGRP receptor antagonist in the treatment of inflammatory pain. Firefly luciferase is commonly used as a reporter in cells expressing a luciferase gene or its enzymatic activity under the control of a promoter of interest to assess its transcriptional activity. It has been found that some molecules such as molecules with carboxylic acid moiety can directly inhibit luciferase activity in cells. However, it is suggested that carboxylic acid moiety of the compounds may also be associated with side reactions in cells. Therefore, to study whether carboxylic acid moiety causes side effects, we designed two probe molecules, 3-1 and 3-2. Synthesis of probe molecule 3-2 is discussed. Synthesis of probe molecule 3-1 and further investigation of its luciferase inhibition will therefore be useful to understand the toxicity of carboxylic acid containing drugs in future.

Les canaux de type T Cav3.2 sont des canaux calciques activés pour de faibles dépolarisations membranaires. Ils ont un rôle important dans la régulation de l’excitabilité neuronale, particulièrement dans les neurones des ganglions rachidiens dorsaux (DRG) où ils sont impliqués dans la transmission de la douleur. Il est établi que les canaux Cav3.2, natifs et recombinants, sont inhibés par de faibles concentrations de métaux divalents tels que le zinc et le nickel et qu’ils sont modulés par des agents oxydo-réducteurs. In vitro, la mutation ponctuelle de l’histidine 191 en glutamine (H191Q) diminue fortement la sensibilité du canal Cav3.2 pour ces différents composés et il est proposé que cette régulation joue un rôle physiologique. L’objectif de ce travail de thèse a été d’étudier l’impact physiologique de cette modulation sur l’excitabilité neuronale et dans la perception de la douleur. Pour ce faire, nous avons généré une souris knock-in (KI) portant la mutation H191Q sur Cav3.2. L’étude électrophysiologique a été réalisée sur une population de neurones de DRG particulière : les cellules D-hair qui sont des mécanorécepteurs exprimant de grands courants Cav3.2. Nous avons validé que la sensibilité des canaux Cav3.2 neuronaux des souris KI est diminuée pour le zinc, le nickel et l’ascorbate. Nous montrons que cette régulation modifiée favorise une augmentation de l’excitabilité de ces neurones. Pour étudier l’impact de cette modulation in vivo, nous avons effectué des études comportementales. Les souris KI ne présentent pas de différence dans la perception de la douleur mécanique et thermique, ni dans l’hyperalgésie induite par l’inflammation et la neuropathie. Toutefois, dans le test à la formaline les souris KI montrent une réponse exacerbée dans la phase tardive. En résumé, nous décrivons ici un modèle animal original pour l’étude de la régulation metal/redox du canal Cav3.2 et identifions un rôle de cette modulation dans l’excitabilité des neurones D-Hair. Nos résultats obtenus in vivo indiquent cependant que cette modulation des canaux Cav3.2 aurait un impact limité dans les voies de la douleur. / Cav3.2 T-type channels are low-voltage activated calcium channels. They have an important role in the regulation of neuronal excitability, particularly in neurons of the dorsal root ganglia (DRG) where they are involved in pain transmission. It is established that Cav3.2 channels are inhibited by low concentrations of divalent metals such as zinc and nickel, and are modulated by redox agents. In vitro, the histidine191-to-glutamine mutation (H191Q) greatly reduces the Cav3.2 channel sensitivity to these compounds and it is proposed that this regulation plays a physiological role. The objective of this thesis was to study the physiological impact of this modulation on neuronal excitability and pain perception. To do this, we generated a knock-in (KI) mouse carrying the H191Q mutation on Cav3.2. Electrophysiological study was carried out on a particular population of DRG neurons, the D-hair cells, which are mechanoreceptors that express large Cav3.2 currents. We show that the sensitivity to zinc, nickel and ascorbate of the neuronal Cav3.2 channels is significantly reduced in the KI mouse. We also show that this modified regulation promotes an increase in the excitability of these neurons. To study the impact of this modulation in vivo, we performed behavioral studies. KI mice show no difference in the perception of mechanical and thermal pain, nor in hyperalgesia induced by inflammation and neuropathy. However, KI mice show an exaggerated response in the late phase in the formalin test. In summary, we describe here an original animal model to study the metal/redox regulation of Cav3.2 channel and identify a role of this modulation in the excitability of D-Hair neurons. Our results indicate, however, that this modulation of Cav3.2 channel may have a limited impact in the pain pathways.