Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn plays a key role in the development and maintenance of pathological pain states and chronic pain diseases. Important role in this process play Transient receptor potential Vanilloid 1 receptors (TRPV1), present on presynaptic endings of primary afferents in the superficial spinal cord dorsal horn. Changes in TRPV1 activity have significant impact on nociceptive transmission. There are number of processes that influence the function of spinal TRPV1 receptors. This work is focused on the role of protease-activated receptors type 2 (PAR2), C-C motif chemokine ligand 2 (CCL2) and the effect of chemotherapeutic drug paclitaxel in modulation of synaptic nociceptive transmission and activation of TRPV1 receptors. PAR2 receptors belong to a family of four G-protein-coupled receptors activated by proteases. The role of PAR2 receptors in pain perception is closely related to their presence in a population of dorsal root ganglion neurons, where they are also co-expressed with TRPV1. Activation of PAR2 may lead to peripheral and central sensitization. Chemokine CCL2 and its main receptor CCR2 were suggested to be an important factor in the development of neuropathic pain after peripheral nerve injury. In our study we focused on the...

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 83-92).

Using patch clamp and Ca2+ imaging techniques, we have studied Ca2+ entry pathways in human hepatoblastoma (HepG2) cells. These cells express the mRNA of TRPV1, TRPV2, TRPV3 and TRPV4 channels, but not those of TRPV5 and TRPV6. Functional assessment showed that capsaicin (10 μM), 4α-phorbol-12,13-didecanoate (4αPDD, 1 μM), arachidonic acid (10 μM), hypotonic stress, and heat all stimulated increases in [Ca2+]i within minutes. The increase in [Ca2+]i depended on extracellular Ca2+ and on the transmembrane potential, which indicated that both driving forces affected Ca2+ entry. Capsaicin also stimulated an increase in [Ca2+]i in nominally Ca2+-free solutions, which was compatible with the receptor functioning as a Ca2+ release channel. Hepatocyte growth factor/scatter factor (HGF/SF) modulated Ca2+ entry. Ca2+ influx was greater in HepG2 cells incubated with HGF/SF (20 ng/ml for 20 h) compared with non-stimulated cells, but this occurred only in those cells with a migrating phenotype as determined by presence of a lamellipodium and trailing footplate. The effect of capsaicin on [Ca2+]i was greater in migrating HGF/SF-treated cells, and this was inhibited by capsazepine. The difference between control and HGF/SF-treated cells was not found in Ca2+-free solutions. 4αPDD also had no greater effect on HGF/SF-treated cells. We conclude that TRPV1 and TRPV4 channels provide Ca2+ entry pathways in HepG2 cells. HGF/SF increases Ca2+ entry via TRPV1, but not via TRPV4. This rise in [Ca2+]i may constitute an early response of a signalling cascade that gives rise to cell locomotion and the migratory phenotype.

Au niveau de la circulation pulmonaire, une exposition prolongée à l’hypoxie est responsable du phénomène de vasoconstriction hypoxique pulmonaire (VHP) qui favorise les échanges gazeux. Lorsque cette VHP se généralise, elle conduit au développement d'une hypertension pulmonaire de groupe 3 (HTP). Cette pathologie se caractérise par un remodelage vasculaire induisant une élévation progressive de la pression artérielle pulmonaire (> 25 mmHg au repos). Ceci conduit à une défaillance cardiaque droite et, à terme, à la mort. La VHP est responsable de l'étirement de la membrane des cellules musculaires lisses des artères pulmonaires (CMLAP) et peut ainsi activer des "Stretch-Activated Channels" tels que les TRPV (Transient Receptor Potential Vanilloid). Il a précédemment été décrit que les canaux TRPV1 et TRPV4, impliqués dans la migration et la prolifération des cellules vasculaires pulmonaires, sont surexprimés et suractivés lors de l'HTP. Cependant, ces modifications peuvent être dues à un effet direct de l’hypoxie ou indirect, conséquence d'un étirement membranaire plus important induit par la VHP. Nous avons donc étudié la contribution respective des stress hypoxique et mécanique, observés en contexte d’HTP, en utilisant des conditionnements in vitro sur des CMLAP d’animaux sains (rats et souris). Nous avons montré que l’hypoxie (1 % O2, 48 heures) induit une augmentation de la [Ca2+]i couplée à une potentialisation de la migration induite par l’activation de TRPV1 et V4. De même, un étirement cyclique (20 %, 1 Hz, 24 heures) provoque une augmentation de la [Ca2+]i et de la prolifération. Ces résultats montrent pour la première fois une action directe de l'hypoxie et du stress mécanique (étirement cyclique) sur des CMLAP. / Hypoxia exposure induces hypoxic pulmonary vasoconstriction (HPV) allowing the efficiency of gas exchanges by increasing the intraluminal pressure. Prolonged hypoxia leads to pulmonary hypertension of group 3 (PH), characterized by increased pulmonary pressure (> 25 mmHg), leading to right ventricular heart failure and ultimately death. HPV leads to stretch pulmonary artery smooth muscle cell (PASMC) membranes inside the vascular wall and thus can activate "Stretch-Activated-Channels" such as TRPV channels (Transient Receptor Potential Vanilloid). It has been previously shown that TRPV1 and TRPV4 channels, implicated in PASMC migration and proliferation, are overexpressed and overactivated in PASMC in the context of PH. But whether this feature is directly caused by hypoxia alone or is a consequence of stretch induced by the HPV is a matter of debate. We thus investigated the respective contribution of hypoxia and mechanical stresses observed in the context of PH using in vitro conditionings on PASMC from healthy animals (rats and mice). We showed that hypoxia (1 % O2, 48 hours) increases Ca2+ entry through TRPV4 channels as well as PASMC migration induced by TRPV1 and TRPV4 activation. Furthermore, cyclic stretch conditioning (20 %, 1 Hz, 24 hours) triggers Ca2+ increase and PASMC proliferation. This work shows for the first time the direct implication of both hypoxia and mechanical stress (cyclic) stretch on PASMC.

Hyperthermia can occur in lungs and airways during both physiological and pathophysiological conditions. A previous study carried out in our laboratory showed that hyperthermia activates and sensitizes vagal bronchopulmonary Cfiber afferents, whether this effect is through a direct action of hyperthermia on sensory nerves is not known. This dissertation study was aimed to investigate the thermal-sensitivity of pulmonary sensory neurons, and the roles of thermalsensitive transient receptor potential vanilloid (TRPV) channels. Whole-cell patch-clamp recordings of neurons isolated from nodose/jugular ganglia were applied in the study. Results of this study showed that hyperthermia directly activates pulmonary sensory neurons, and this effect is partially mediated through the TRPV subtype 1 (TRPV1) channel as well as other thermal-sensitive TRPV (2–4) channels. In addition, hyperthermia exerts potentiating effects on responses of pulmonary sensory neurons to TRPV1 activators, but not to non- TRPV1 activators. Furthermore, results obtained in the study of TRPV1-null mice revealed that TRPV1 plays a dominant role in mediating the potentiating effect of hyperthermia on pulmonary sensory neurons, but is only partially involved in the direct activation of these sensory neurons by increasing temperature. These results suggested that the thermal-sensitivity of pulmonary sensory neurons is dependent upon the function of the TRPV1 channel, and TRPV1-mediated sensitization of these sensory neurons may contribute to airway hyperreactivity and augmented reflex responses under hyperthermic conditions.

Membrane proteins live at the interface between a cell and its environment; hence, they play a variety of important physiological roles such as transmembrane transport, signal transduction, and cell adhesion. The importance of membrane proteins in biology and medicine requires that we understand their structure and function on the atomic level. In this thesis, I studied members of two different membrane protein families, namely the neuronal and keratinocyte TRPV ion channels that sense temperature changes and MP20, a member of the PMP22/EMP/MP20/claudin superfamily. Using a variety of biochemical, X-ray crystallographic and electrophysiological techniques, I addressed mechanistic questions pertaining to the regulation of thermosensitive TRPV channels by ATP and calmodulin in neurons and keratinocytes. For MP20, a protein specific for the lens of the mammalian eye, I used a vesicle assay in combination with electron microscopy (EM) to study its function, ruling out the possibility that MP20 is involved in the formation of membrane junctions. Furthermore, I made progress in expressing and crystallizing MP20 for X-ray diffraction studies. In a separate effort, I also worked on improving and expanding the use of monolayer purification and Affinity Grids, recently introduced techniques to prepare specimens for single-particle EM based on the recruitment of His-tagged proteins to nickel lipidcontaining lipid monolayers. I extended the use of these techniques by synthesizing a glutathione lipid that can be used to recruit GST-tagged proteins. A major hurdle in the use of monolayer purification techniques, however, is the extent of non-specific protein binding to the lipid monolayer. I found that incorporating PEG lipids in the monolayer appears to reduce the problem of non-specific protein binding. While it remains to be seen whether these techniques can be developed to a point at which it will be possible to recruit exclusively tagged proteins out of cell lysates, my goal is to continue to improve and expand the use of the monolayer purification and Affinity Grid techniques in hope to make single-particle EM more easily amenable to biochemists and cell biologists.

Splice variants of the transient receptor potential vanilloid type-1 (TRPV1) channel appear to be involved in the physiological detection of extracellular fluid (ECF) osmolality in the supraoptic nucleus (SON) and organum vasculosum lamina terminalis (OVLT). It remains to be determined whether these splice variants are directly involved as pore-forming proteins in the osmosensory transduction complex. Since these TRPV1 splice variants are not sensitive to capsaicin antagonists, such as capsazepine (Sharif Naeini et al., 2007), novel tools that specifically interfere with ion permeation through TRPV1 are required for functional studies on the involvement of this channel. In this study, we developed rabbit polyclonal antibodies targeting specifically the extracellular pre-pore loop region of rat TRPV1 (PH-4281). Histological results showed that PH-4281 is specific to rat TRPV1 and TRPV1 expression is found in regions that are known to be osmosensitive. PH-4281 could be used as a specific tool to study the osmosensory transduction complex.

No description available.

<p>For all organisms, the ability to sense and react to noxious environments is fundamental to their survival. For multi-celled organisms this process generally involves a nervous system and an extensive network of signal transduction pathways. TRPV ion channels have been shown to participate in signal transduction in response to noxious stimuli. At the cellular level these channels function in sensing of mechanical, thermal, and osmotic stimuli, and at the organismal level they function in homeostasis and nociception. TRPV ion channels participate in nociceptive signal transduction via cation influx, but exactly how these channels function at a mechanistic level and lead to activation of the cell or induction of a specific behavior is elusive. Previous research has shown that the pore-forming unit of an ion channel is critical for channel regulation, gating, ion selectivity, and ion permeation. Various regulatory domains have been identified to date in the pore-forming unit of TRP channels and a clearer picture of channel gating is beginning to emerge, but less is known about ion permeation. </p><p>To better understand the specific domains that are critical to ion capture, selectivity, and permeation in TRPV channels, we investigated the function of these regions using the <italic>C. elegans</italic> TRPV channel OSM-9 <italic>in vivo</italic>, and the mammalian TRPV channel TRPV4 in heterologous cell culture. OSM-9 is the functional ortholog of mammalian TRPV4 and it is likely that critical domains identified in OSM-9 are functionally conserved in TRPV4 and play a similar role in other TRPV channels. OSM-9 is expressed in the ASH neurons and is responsible for all of the behaviors initiated by that cell. The stereotypical avoidance behavior mediated by ASH, in response to noxious stimuli, serves as a model for nociception in vertebrates. As OSM-9 is necessary for all of these behavioral responses, activation of ASH acts as a read-out for OSM-9 function.</p><p>Through targeted mutagenesis of the OSM-9 loop domains and transgenic expression directed to the ASH head sensory neurons in an <italic>osm-9</italic> null background, we discovered a critical role for the amino acids both N- and C- terminal to the pore helix in osmotic avoidance behavior. We confirmed the existence of a selectivity filter C-terminal to the pore helix and revealed that the turret is critical for channel function, possibly as a component of the inactivation gate.</p><p>We first identified the boundaries of the selectivity filter to be M601-F<super>609</super>. We also determined what properties of those residues were critical to Ca<super>2+</super> and Na<super>+</super> selectivity. <italic>In vivo</italic> Ca<super>2+</super> imaging strongly suggested that residues Y<super>604</super>, D<super>605</super>, and F<super>609</super> are critical for Ca<super>2+</super> entry into the cell. Patch-clamp electrophysiology of a chimeric ion channel consisting largely of rat TRPV4, but encompassing transmembranes 5 through 6 of OSM-9, revealed that OSM-9 conducts both Ca<super>2+</super> and Na<super>+</super>. Mutation Y604G disrupted both Ca<super>2+</super> and Na<super>+</super> conductance, whereas mutations Y604F and Y606A increased or maintained Na+ conductance and severely reduced Ca<super>2+</super> conductance, while maintaining avoidance behavior. Homology modeling of OSM-9, based on an alignment of OSM-9 to Kv1.2, suggests that Y<super>604</super> and F<super>609</super> serve structural roles in maintaining filter constraints. Thus, aromatic and negative residues in the OSM-9 selectivity filter are critical to ion permeation and selectivity. </p><p>Our studies involving the selectivity filter support previous research that the selectivity filter is critical for TRP channel function. We also provide evidence that the selectivity filter is critical for nocifensive animal behavior. Fewer studies, however, have investigated the TM5-pore helix linker, known as the turret. The turret is believed to function in the binding of ligands and toxins in K<super>+</super> channels, and more recently was suggested to be critical for temperature sensing in TRPV1. We investigated the function of the turret residues in several sensory submodalities of the OSM-9 channel and found that all deletions tested result in channel defects, including gain- and loss-of-function phenotypes. Several charge reversal mutations in the OSM-9 turret also resulted in partial defects. The discovery of a gain-of-function mutation indicates that the turret functions in gating. When the turret is mutated in this way, the channel is unable to enter into the inactivated state, allowing continued ion influx after repeated stimulation. The loss-of-function phenotypes indicate that the secondary structure of the turret is critical to the function of the channel, and perhaps gating. These findings, combined with the observed charge-reversal defects, support the conclusion that the turret is necessary for transducing conformational changes in response to stimuli.</p><p>Our <italic>in vivo</italic> findings on the external pore forming structures increase the understanding of ion permeation in TRP channels and clarify mechanisms of activation in nociceptor neurons <italic>in vivo</italic>. Furthermore, these studies enhance our insights into evolution of mammalian nociception in view of the established functional orthology of OSM-9 and TRPV4.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (p. 73-82).