Le succès de la réponse immunitaire repose en grande partie sur la capacité des leucocytes à se déplacer et à accomplir leur fonction au sein de structures anatomiques précises. Le fait qu’il puisse exister des mécanismes intrinsèques de coordination entre ces fonctions spécifiques et la migration de ces cellules n’a jamais été étudié auparavant. Nos travaux mettent en évidence, pour la première fois, l’existence d’un couplage entre la migration et la macropinocytose dans les cellules dendritiques qui explorent leur environnement en internalisant une grande quantité de matériel extra-cellulaire. C’est la Chaîne Invariante, protéine chaperon impliquée dans l’apprêtement des antigènes, qui est responsable de ce couplage en détournant le moteur Myosine II de l’arrière de la cellule, où elle promeut la migration, vers l’avant de la cellule. Ce recrutement transitoire de Myosin II autour des macropinosomes à l’avant favorise la macropinocytose et la délivrance de l’antigène dans les lysosomes, mais ralentit la cellule. L’implication de la Myosine II à la fois dans la migration et la capture d’antigène permet donc le couplage moléculaire entre ces deux processus et leur coordination spatio-temporelle. Cependant, les voies de signalisation impliquées dans le couplage avant/arrière dans les cellules dendritiques immatures restent encore méconnues. L’ensemble de mes travaux de thèse montrent que la libération de calcium du réticulum endoplasmique à travers les récepteurs IP3 (IP3Rs) est nécessaire pour maintenir le niveau de phosphorylation de la chaîne légère de Myosin (MLC) et la polarisation avant/arrière de Myosine II au cours de la migration des cellules dendritiques immatures. Nous montrons que les récepteurs IP3R1, 2 et 3 sont requis pour atteindre une vitesse maximale en 2- et 3-Dimension, et que le récepteur IP3R3, et dans une moindre mesure IP3R1, favorisent la persistance des cellules. En revanche, l’inhibition de l’expression du récepteur IP3R3 augmente la capacité des cellules dendritiques immatures à capturer l’antigène, ce qui est en accord avec notre résultat montrant que la capture de l'antigène est inversement reliée à la locomotion de cellules dendritiques. Nous proposons que le relargage du calcium par le réticulum endoplasmique favorise l’activité de la myosine II ce qui permet aux cellules dendritiques de ralentir de façon transitoire. Ce relargage calcique permet aux cellules dendritiques du optimiser l'internalisation des antigènes extracellulaires en maintenant leur polarité ce qui leur permet d’optimiser ainsi leur capacité d'échantillonnage de l’environnement. / The immune response heavily relies on the migration capacity of leukocytes. These cells must stop in precise anatomical locations to fulfill a particular task. But whether and how specific functions are coordinated with migration by cell-intrinsic mechanisms is not known. We here show that in dendritic cells, which patrol their environment for the presence of antigens by internalizing extracellular material, macropinocytosis is coupled to cell migration. Coupling relies on the diversion of the Myosin II motor from its migratory function at the cell rear to macropinosomes at the cell front by the Invariant Chain, a cell-specific regulator of antigen presentation. Transient Myosin II recruitment at the cell front promotes antigen macropinocytosis and antigen delivery to endolysosomes but antagonizes cell migration. Thus, the requirement for Myosin II for both migration and antigen capture provides a molecular mechanism to couple these two processes and allow their coordination in time and space. However, the signaling pathways involved in back/front coupling in migrating immature DCs remain unknown. Here we show that calcium released from the endoplasmic reticulum through IP3 Receptors (IP3Rs) is required to maintain Myosin regulatory light Chain (MLC) phosphorylation and Myosin II back/front polarization during DC locomotion. We found that while IP3R1, 2 and 3 are required for immature DCs to reach maximal speed in 2-Dimensional and 3-Dimensional environments, IP3R3 and to a lesser extent IP3R1 positively regulate their persistency. On the contrary, silencing of IP3R3 increases antigen uptake by immature DCs, consistent with our finding showing that antigen capture is inversely coupled to DC locomotion (Appendix, manuscript #1). We propose that by promoting myosin II activity, calcium released from the ER help DCs to transiently slow-down to uptake extracellular antigens without losing their polarity and thereby optimizes their environment sampling capacity.

Dans les synapses chimiques, des canaux calciques voltage-dépendants (VDCC) provoquent la fusion des vésicules synaptiques (SV) au niveau de la zone active. L’efficacité et la rapidité de la transmission synaptique dépendent de la distribution relative entre les VDCCs et les SVs prêtes à fusion. Cependant, les modalités d’interaction entre les VDCCs et les SVs ne sont pas connues. Afin de localiser les VDCCs à l’échelle nanométrique j’ai developpé une nouvelle approche chez Caenorhabditis elegans combinant le marquage in vivo des VDCCs, grâce à l’expression d’un épitope extracellulaire, et la microscopie électronique (EM). J’ai généré un transgene GFP::unc-36 qui code la seule sous-unité α2-δ qui s’associe à fois avec les sous-unités formant le pore α1 neuronal (UNC-2) et musculaire (EGL-19) chez C.elegans. J'ai ensuite utilisé des quantum dots conjugués avec l’anticorps anti-GFP, fluorescents et denses au électrons, pour localiser des VDCCs à haute résolution au niveau de la jonction neuromusculaire (NMJ) par EM. En parallèle, j'ai utilisé la technique de CALM (complementation activated light microscopy) pour étudier la dynamique des VDCC dans des vers vivants. Nos résultats montrent que les VDCCs diffusent à l’échelle de nanodomaines sur la membrane musculaire. De plus leur diffusion est modulée en réponse à la tension musculaire. La dystrophine participe au couplage électro-mécanique au niveau du sarcolemme en modulant la taille du domaine de confinement des VDCCs. Enfin, nous avons mis en evidence le rôle de RIM/UNC-10 dans la régulation de la mobilité latérale des VDCCs dans les neurones, probablement via son interaction avec les VDCCs et les SVs. / At chemical synapse voltage-dependent calcium channels (VDCC) trigger synaptic vesicles (SV) fusion at the active zone in response to depolarization stimuli. Intracellular Ca2+ influx forms a nanodomain around individual VDCC. Fast and efficient synaptic transmissions appear to be tightly coupled with the relative distribution between the VDCCs and SVs fusion sites. However, the connection between VDCCs and docked SVs at a few nanometer scales remain enigmatic. To localize VDCCs in nanometer resolution I developed a novel approach combining in vivo labeling of VDCCs via genetically-encoded extracellular epitope tags and electron microscopy (EM). I engineered a GFP/split-GFP tag fused at the extracellular N-terminal of UNC-36, the only C. elegans VDCC α2δ subunit associating with both neuronal (UNC-2) and muscular (EGL-19) VDCC pore-forming α1 subunit. I then used quantum dot (QD) conjugated antibodies as both fluorescent and electron dense probes to localize VDCCs at C. elegans neuromuscular junction (NMJ) by in vivo QD-antibodies labeling and EM. In parallel, I applied in vivo complementation activated light microscopy to study VDCC dynamics in live worms. I discovered that VDCCs diffuse within nanodomains at sarcomeric membrane and their nanoscale diffusion behavior is modulated in response to muscle tension. In addition, we found that dystrophin participates in electro-mechanical coupling at the sarcolemma by modulating the confinement size of VDCCs. Meanwhile, we discovered lateral mobility of N-type VDCC at NMJs, and that RIM/UNC-10 seems involved in regulation of VDCC dynamics via its interaction with VDCC and SVs.

This thesis details my examination of several mechanisms for modulation of N-type calcium channels in neonatal rat superior cervical ganglion (SCG) neurons. The first part of this work characterizes cross-talk between two distinct mechanisms of modulation: readily-reversible inhibition induced by activation of heterotrimeric G-proteins (termed G-protein-mediated inhibition), and phosphorylation of the channel by protein kinase C (PKC). Data previously presented by other groups suggested that one effect of activating PKC is to prevent G-protein-mediated inhibition. The goal of this project was to confirm this hypothesis by testing functional competition between these two pathways. My findings show that G-protein-mediated inhibition blocks the effects of activating PKC, and that phosphorylation by PKC blocks G-protein-mediated inhibition, confirming that these two mechanisms are mutually exclusive. In addition, I investigated the effect of activating PKC on whole-cell barium currents in the absence of G-protein-mediated inhibition. When endogenous G-proteins were inactivated by dialyzing the cell with GDP-β-S, a guanine nucleotide that prevents activation of the G-protein's α subunit, activation of PKC with phorbol esters was without obvious effect on whole-cell current amplitude, fast and holding potential-dependent inactivation, and voltage-dependent activation, suggesting that PKC's principal role in modulating these currents is to prevent G-protein-mediated inhibition. From these results, I advanced Bean's 1989 model of reluctant and willing gating (induced by G-protein-mediated inhibition and relief of that inhibition, respectively). In this expanded model, reluctant channels, inhibited by G-proteins, are resistant to phosphylation by PKC (reluctant/P-resistant). Unmodulated channels are called willing/available, as they exhibit willing gating, and are available for either binding to a G-protein or phosphorylation by PKC. Finally, phosphorylation of a willing/available channel by PKC drives the channel into the willing/G-resistant state, in which the channel gates willingly, and is resistant to G-protein-mediated inhibition. These results are published in the Journal of General Physiology(2000; 115:277-286), and are presented in this thesis as Chapter II. In addition to membrane-delimited inhibition, N-type calcium channels are also subject to inhibition via a diffusible second-messenger pathway. In SCG neurons, this inhibition can be observed following stimulation of M1 muscarinic receptors by the agonist oxotremorine-M. Our lab previously hypothesized that the diffusible messenger involved might be the polyunsaturated fatty acid arachidonic acid (AA). To test this hypothesis, our lab examined the effect of bath-applied AA on whole-cell SCG calcium currents, and demonstrated that AA induces inhibition with similar properties as M1 muscarinic inhibition. An analysis of AA's effects on unitary N-type calcium currents, published by Liu and Rittenhouse in Journal of Physiology(2000; 525:391-404), revealed that this inhibition is mediated, at least in part, by both a significant increase in the occurrence of null-activity sweeps and a significant decrease in mean closed dwell time. Based on these results, our lab conducted an examination of AA's effects on whole-cell currents in SCG neurons, and found that AA-induced inhibition is mediated by an increase in holding potential-dependent inactivation and appears independent of AA metabolism. When I examined AA's effects in greater detail, I discovered that, in addition to inhibition, AA also appeared to cause significant enhancement of whole-cell currents. The results characterizing AA's general effects on whole-cell calcium currents in SCG neurons have been published in American Journal of Physiology - Cell Physiology(2001; 280:C1293-C1305). Because my finding that AA enhances whole-cell neuronal calcium currents revealed a novel pathway through which this current can be modulated, I proceeded to characterize this effect. My results showed that enhancement develops significantly faster than inhibition, suggesting different mechanisms or pathways. In addition, dialyzing the cell with BSA, a protein that binds fatty acids, blocked the majority of AA-induced inhibition, but did not reduce enhancement, suggesting that enhancement is independent of inhibition and might be mediated at an extracellular site. Using fatty acid analogs that cannot cross the cell membrane, I confirmed that enhancement occurs extracellularly. My data also indicate that AA-induced enhancement of whole-cell currents does not require metabolism of AA, consistent with enhancement being mediated directly by AA. I also examined the biophysical characteristics of enhancement, and found that both an increase in the voltage sensitivity of activation and an increase in activation kinetics underlie this effect. Finally, using both pharmacological agents and a recombinant cell line, I presented the first demonstration that AA enhances N-type calcium current. These findings are described in detail in a paper recently published in American Journal of Physiology - Cell Physiology(2001; 280:C1306-C1318), and are presented in this thesis as Chapter III. In our investigation of AA's effects on whole-cell calcium currents, we utilized a voltage protocol, in conjunction with pharmacology, to enhance the level of L-type current in these cells. Since whole-cell calcium currents in SCG neurons are comprised of mostly (80-85%) N-type current, with the remaining current comprised of mostly L-type current, this approach allowed us to examine both N- and L-type currents. When currents are recorded in the presence of 1 μM FPL 64174 (FPL), a benzoyl pyrrole L-type calcium channel agonist first described in 1989, stepping the membrane potential to -40 mV following a test pulse to +10 mV generates a slowly-deactivating ("tail") current. This tail current is made up entirely of L-type current, and allows us to readily investigate the effect of various modulatory mechanisms on this current type. Although FPL has been used for almost a decade to study L-type calcium currents, activity of FPL on N-type calcium currents has not been investigated. Because our lab routinely uses micromolar concentrations of FPL to measure whole-cell and unitary calcium currents in neuronal cells, I tested whether FPL has any effects on N-type calcium current. Therefore, I examined the effect of FPL on whole-cell calcium currents in an HEK 293 cell line that expresses recombinant N-type calcium channels. Application of 1 and 10 μM FPL caused significant, voltage-independent inhibition of currents, demonstrating that FPL inhibits N-type calcium current. Thus, at micromolar concentrations, FPL is not selective for L-type calcium current, and any examination of its effects on whole-cell calcium currents should take this into account. The results describing FPL's effects on L- and N-type calcium currents are included in a manuscript currently in preparation, and are presented as Chapter IV.

This thesis details my examination of several mechanisms for modulation of neuropeptide release via voltage-dependent and voltage-independent intraterminal signaling in isolated neurohypophysial terminals. The first part of this work characterizes depolarization-induced neuropeptide release in the absence of extracellular calcium. The goal of this project was to examine the relationship between depolarization-induced release of intracellular calcium stores and depolarization-secretion coupling of neuropeptides. We demonstrate that depolarization in the absence of extracellular calcium induced by either High K+ or electrical stimulation induces a rise in [Ca2+]i and subsequent neuropeptide release from Hypothalamic Neurohypophysial System (HNS) terminals. A portion of extracellular calcium-independent neuropeptide release is due to intraterminal calcium, but the remaining depolarization-induced release may be due to calcium-independent voltage-dependent (CIVD) release (Zhang and Zhou, 2002; Zhang et al., 2004; Yang et al., 2005). Nevertheless, our results clearly show that extracellular calcium is notnecessary for depolarization-induced neuropeptide secretion from these CNS terminals. In addition, I investigated the role of internal calcium stores in mediating μ-opioid inhibition of voltage-gated calcium channels (VGCCs). Inhibition of VGCCs via μ-opioid agonists has been shown to reduce neuropeptide release in response to High K+ stimulation of isolated terminals (Bicknell et al., 1985b; Russell et al., 1993; van Wimersma Greidanus and van de Heijning, 1993; Munro et al., 1994; Ortiz-Miranda et al., 2003; Russell et al., 2003; Ortiz-Miranda et al., 2005). My findings show μ-opioid inhibition, of VGCC and High K+-mediated rise in [Ca2+]i, are via a voltage-independent diffusible second-messenger targeting release of calcium from ryanodine-sensitive stores, possibly mediated via the cyclic ADP ribose signaling pathway. Furthermore, I detail a different intracellular messenger pathway mediating the κ-opioid inhibition of VGCC and High K+-mediated rise in [Ca2+]ii. In contrast to the μ-opioid inhibition, κ-receptor activation is coupled to a voltage-dependent membrane-delimited pathway. Inhibition of neuropeptide release via both endogenous and exogenous κ-opioid agonists has been extensively studied (Bicknell et al., 1985a; Nordmann et al., 1986a; Wammack and Racke, 1988; Munro et al., 1994; Ingram et al., 1996; Rusin et al., 1997a). My investigation shows that the κ-inhibition of VGCC is voltage-dependent and is furthermore, relieved within the context of a physiological burst of action potentials (APs). This physiologically-evoked, activity-dependent modulation of VGCC and subsequent release, represents an important mechanism for short-term synaptic plasticity at the level of the terminals. Given the ubiquitous nature of voltage-dependent G-protein signaling in the CNS, our results may prove important in understanding modulatory effects of specific bursting patterns throughout the CNS. In the last 30 years the neurohypophysial system has proven to be an excellent system to study the complexities of depolarization-secretion coupling (DSC). There have been many advances in our understanding of the underlying mechanisms involved and their physiological implications. The current work focuses on two important features of DSC; voltage and calcium. Although in many ways these two are intrinsically linked through VGCC activation, we have found that in isolated HNS terminals that is not always the case. We have further found that when voltage and calcium influx are linked during DSC, modulation by opioids may or may not be linked to activity-dependent relief depending on the opioid receptor activated. This finding has important implications in neuropeptide release during patterned stimulation in vivo. As I will discuss further, many factors play into the complexities of the regulatory mechanisms involving release. As investigations into this remarkable field continue, I hope to have contributed a valuable piece to the puzzle.

Membrane excitability, gene expression, and neurotransmitter release are all controlled by voltage-gated L-type Ca2+ (L- )channels. In turn, Ca2+ channels are highly regulated by signal transduction cascades initiated by G protein-coupled receptor (GPCR) activation. In medium spiny neurons of the striatum, both the muscarinic M1 receptors (M1R) and dopaminergic D2 receptors (D2R) specifically inhibit the Cav1.3 L-channel. In Chapters III and IV, the pathways downstream of M1Rs and D2Rs are examined to determine whether an overlap or intersection in inhibition of Cav1.3 occurs by these two receptors. Transient transfection of Cav1.3 channels in HEK 293 cells, stably transfected with the M1R, and in ST14A cells were used as model systems. While a further characterization of ST14A cells determined that they exhibit a striatal profile, D2Rs or M1Rs did not inhibit Cav1.3. Lack of current inhibition may be due to the finding of no detectable expression of phospholipase Cβ-1 protein in ST14A cells. Ca2+ channels are multiprotein complexes comprised of α1, β, and α2δ subunits. While the actions of arachidonic acid (AA) have been shown to mimic M1R inhibition of L-current in superior cervical ganglion neurons, the precise identity of the L-channel in these neurons -either Cav1.2 or Cav1.3 or both- is not known. The transfected model systems allowed for the analysis of whole-cells currents with different β subunit combinations as well as the study of only Cav1.3 channels. In Chapter III, I show that activation of M1Rs with the agonist Oxo-M inhibited Cav1.3 channels coexpressed with either β1b, β2a, β3, or β4 subunits. Surprisingly, the magnitude of Cav1.3, β2a currents was inhibited less than Cav1.3 currents with other β subunits. In Chapter V, AA is shown to mimic the profile of M1R stimulation on Cav1.3 currents. The magnitude of Cav1.3, β2a currents was inhibited less than Cav1.3 currents with other β subunits by AA. This discovery points to a novel role for accessory β subunits in altering the magnitude of AA inhibition and kinetic changes of Cav1.3. Arachidonic acid (AA) inhibits Ca2+ channels by an unknown mechanism at an unknown site. In Chapter V, I found that Cavl.3 inhibition by AA was state-dependent and most likely stabilizes a closed channel conformation. The finding that the Ca2+ channel accessory β subunit alters the magnitude of AA inhibition and kinetic changes of Cav1.3 suggests that AA could alter processes which rely on L-channels such as Ca2+-dependent gene expression, secretion and membrane excitability.

The influx of calcium through N-type calcium channels (N-current) affects a myriad of neuronal functions. These include the triggering of synaptic release of neurotransmitter, adjustment of membrane potential and changes in gene transcription. N-channels are highly modulated proteins, so that N-current is attenuated or potentiated in response to environmental changes. In turn, the modulation of N-current has a direct effect on the downstream events, making the N-channel a focal point in neural signaling, and its modulation a mechanism for short term plasticity. The modulation of N-current by M1 muscarinic receptors (M1Rs) is of particular interest for several reasons. The M1R is instrumental in both cognition and memory formation as indicated by studies using either pharmacological agents aimed at M1Rs or knockout animals lacking M1Rs. Clinically, the M1R is an important target in the treatment of Alzheimer’s disease. Thus, like the N-channel, the M1R is an important element of neural signaling. Moreover, the stimulation of M1Rs affects N-current by through signaling pathways which despite being studied for decades, are not completely understood. For my dissertation I have investigated of M1R signaling on N-current using electrophysiological recordings of N-current from freshly dissociated neurons and from HEK cells expressing N-channels and M1Rs. Asking how one receptor affects one type of calcium channel would seem to be a simple question. However, the answer has many facets. Since M1Rs have multiple downstream effects and N-channels are highly modulated proteins, stimulation of M1Rs initiates several different pathways which modulate N-current. This thesis aims to unravel some of the complexities of the interactions of two vital components of neuronal signaling. Here I present the results of studies elucidating three different actions of M1signaling of N-current modulation. The first study I present here examines the effect of N-channel subunit composition on modulation of N-current. The stimulation of M1Rs in superior cervical ganglion (SCG) neurons elicits a distinct pattern of modulation; inhibiting N-current elicited by strong depolarizations and enhancing current elicited by lesser depolarizations. Thus M1Rs cause two simultaneous modulatory effects on N-current; increasing voltage sensitivity and decreasing overall conductance. I found the expression of the N-channel’s β subunit (CaVβ) determines the observed effect. Specifically when the isoform CaVβ2a is expressed M1 stimulation elicits enhancement without inhibition. Conversely, when CaVβ1b, CaVβ3, or CaVβ4 are expressed M1 stimulation elicits inhibition with out enhancement. These results fit a model in which both the enhancing and inhibiting effects of M1stimulation occur in all channels, but typically inhibition dominates. CaVβ2a blocks inhibition unmasking latent enhancement. Moreover, using mutants and chimeras I found palmitoylation of CaVβ2a at the N-terminus plays a key role in blocking inhibition. My findings predict the expression and localization of different CaVβ isoforms would dramatically alter modulation of N-current and thus may represent a previously unrecognized form of plasticity. The inhibition of N-current by M1Rs is controversial. It has been proposed recently that inhibition is directly attributable to the depletion of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during M1 stimulation. However, in our lab, we have found arachidonic acid (AA) release, which occurs subsequent to PtdIns(4,5)P2 hydrolysis, is both necessary and sufficient to elicit inhibition. Therefore, in a second study, I tested the effect of CaVβ expression on N-current during exogenous AA application and found a pattern of modulation identical to M1R stimulation. Furthermore, I took part in a collaborative project identifying the AA producing enzyme, diacylglycerol lipase (DAGL), to be a necessary component of the inhibitory pathway elicited by M1Rs. These findings provide increased evidence for AA release being a key factor in the M1R stimulated pathway of inhibition. Moreover, these discoveries identify the expression of CaVβ2a and use of specific DAGL inhibitors as a molecular and pharmacological strategy to block inhibition of N-current, respectively. These tools allow the dissection of downstream effects of M1R stimulation, so that other modulatory effects may be observed. The phosphorylation of N-channels by protein kinase C (PKC) blocks inhibition of current brought on by G-protein β and γ subunits (Gβγ) binding directly to the channel. Relief of Gβγ inhibition by other means has been identified as a mechanism of short term plasticity. M1Rs are known to simulate PKC, but a connection between M1Rs and PKC phosphorylation of Nchannels had not been demonstrated. I hypothesized that PKC stimulation may be occluded by other downstream effects of M1Rs. Therefore in a third study, I used a pharmacological approach on SCG neurons to dissect the PKC activating pathway from the other downstream effects of M1 stimulation. I observed modulation of N-current indicating a loss of Gβγ&#; inhibition, thus consistent with PKC phosphorylation of channels. This conclusion reveals another aspect of M1 modulation, which can function as a means of short term plasticity.

Indiana University-Purdue University Indianapolis (IUPUI) / Protein-protein interactions play a crucial role in a wide range of biological processes. Research on the design and synthesis of small molecules to modulate these proteinprotein interactions can lead to new targets and drugs to modulate their function. In Chapter one, we discuss the design and synthesis of small molecules to probe a proteinprotein interaction in a voltage-gated Ca2+ channel. Virtual screening identified a compound (BTT-3) that contained a 3,4-dihydro-3,4’-pyrazole core. This compound had modest biological activity when tested in a fluorescence polarization (FP) assay. The synthetic route to BTT-3 consisted of six steps. In addition, analogs of BTT-3 were made for a structure-activity study to establish the importance of a carboxylate moiety. We also synthesized a biotinylated benzophenone photo-affinity probe and linked it to BTT-3 to identify additional protein targets of the compound. In Chapter two, small-molecule antagonists targeting uPA-uPAR protein-protein interaction are presented. A total of 500 commercially-available compounds were previously identified by virtual screening and tested by a FP assay. Three classes of compounds were found with biological activity. The first class of compounds contains pyrrolidone core structures represented by IPR- 1110, the second class has a novel pyrrolo[3,4-c]pyrazole ring system, represented by xv IPR-1283 and the last series had compounds with a 1,2-disubstituted 1,2- dihydropyrrolo[3,4-b]indol-3(4H)-one core structure, represented by IPR-540. Each of these three compounds were synthesized and assessed by FP and ELISA assays. A binding mode of IPR-1110 with uPA was subsequently proposed. Based on this binding mode, another 61 IPR-1110 derivatives were synthesized by us to illustrate the SAR activity. Analogs of the other two series were also synthesized.

Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channel Nav1.7 has gained traction as a pain target with recognition that loss-of-function mutations in SCN9A, the gene encoding Nav1.7, are associated with congenital insensitivity to pain, whereas gain-of-function mutations produce distinct pain syndromes due to increased Nav1.7 activity. Selective inhibition of Nav1.7 is fundamental to modulating pain via this channel. Understanding the regulation of Nav1.7 at the cellular and molecular level is critical for advancing better therapeutics for pain. Although trafficking of Nav1.7 remains poorly understood, recent studies have begun to investigate post-translational modifications of Navs and/or auxiliary subunits as well as protein-protein interactions as Nav-trafficking mechanisms. Here, I tested if post-translational modifications of a novel Nav1.7-interacting protein, the axonal collapsin response mediator protein 2 (CRMP2) by small ubiquitin-like modifier (SUMO) and phosphorylation could affect Nav trafficking and function. Expression of a CRMP2 SUMOylation incompetent mutant (CRMP2-K374A) in neuronal model CAD cells, which express predominantly Nav1.7 currents, led to a significant reduction in huwentoxin-IV-sensitive Nav1.7 currents. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wildtype CRMP2-expressing CAD cells decreased Nav1.7 currents. Consistent with reduced current density, biotinylation revealed significant reduction in surface Nav1.7 levels of CAD cells expressing CRMP2-K374A or SENP proteins. Diminution of Nav1.7 sodium current was recapitulated in sensory neurons expressing CRMP2-K374A. Because CRMP2 functions are regulated by its phosphorylation state, I next investigated possible interplay between phosphorylation and SUMOylation of CRMP2 on Nav1.7. Phosphorylation of CRMP2 by cyclin dependent kinase 5 (Cdk5) was necessary for maintaining Nav1.7 surface expression and current density whereas phosphorylation by Fyn kinase reduced CRMP2 SUMOylation and Nav1.7 current density. Binding to Nav1.7 was decreased following (i) loss of CRMP2 SUMOylation, (ii) loss of CRMP2 phosphorylation by Cdk5, or (iii) gain of CRMP2 phosphorylation by Fyn. Altering CRMP2 modification events simultaneously was not synergistic in reducing Nav1.7 currents, suggesting that Nav1.7 co-opts multiple CRMP2 modifications for regulatory control of this channel. Loss of either CRMP2 SUMOylation or Cdk5 phosphorylation triggered Nav1.7 internalization involving E3 ubiquitin ligase Nedd4-2 as well as endocytosis adaptor proteins Numb and Eps15. Collectively, my findings identify a novel mechanism for regulation of Nav1.7.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: During glutamate excitotoxicity, overstimulation of glutamate receptors leads to sustained elevation in cytosolic Ca2+ ([Ca2+]c), or delayed Ca2+ dysregulation (DCD), which is causally linked to cell death. There are two major hypothetical mechanisms for DCD: the continuous activation of N-methyl-D-aspartate-subtype of the ionotropic glutamate receptors (NMDAR) and the reversal of the plasmalemmal Na+/Ca2+ exchanger. However, the contribution of each of these mechanisms in DCD is not completely established. Major results: Neurons exposed to excitotoxic glutamate produced DCD, an increase in cytosolic Na+ ([Na+]c), and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added after glutamate, completely prevented DCD; however AP-5, a competitive NMDAR inhibitor, failed to do so. The NMDAR inhibitors had no effect on lowering elevated [Na+]c or on restoring plasma membrane potential, which are conditions suggesting NCXrev could be involved. In experiments inducing NCXrev, MK801 and memantine completely inhibited Ca2+ dysregulation after glutamate while AP-5 did not. Inhibition of NCXrev, either with KB-R7943 or by preventing the increase in [Na+]c, failed to avert DCD. However, NCXrev inhibition combined with NMDAR blocked by AP-5 completely prevented DCD. Overall, these data suggested that both NMDAR and NCXrev are essential for glutamate-induced DCD, and inhibition of only one mechanism is insufficient to prevent collapse of calcium homeostasis. Based on the data above, we investigated a NMDA receptor antagonist currently in clinical trials for reducing the effects of glutamate excitotoxicity, ifenprodil. Ifenprodil is an activity-dependent, NMDAR inhibitor selective for the NR2B subunit. We found that ifenprodil not only inhibited the NR2B-specific NMDAR, but also inhibited NCXrev. If ifenprodil is combined with PEAQX, a NMDAR inhibitor selective for the NR2A subunit, low concentrations of both inhibitors completely prevent DCD. Conclusion: The inhibition of a single Ca2+ influx mechanism is insufficient in preventing DCD, which requires simultaneous inhibition of both the NMDAR and NCXrev. These findings are critical for the correct interpretation of the experimental results obtained with these inhibitors and for better understanding of their neuroprotective actions.

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