Thesis (M.S.)--Duquesne University, 2009. / Abstract included in electronic submission form. Title from document title page. Includes bibliographical references (p. 65-71) and index.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 157-188).

No description available.

Transient receptor potential (TRP) channels are expressed throughout the central nervous system and have a unique ability to detect a wide range of stimuli including changes in voltage, temperature, pH, lipid environment, small molecule agonists, and mechanical stress. While it is known that TRP channels contain the same six transmembrane helix (S1-S6), tetrameric architecture as voltage-gated channels, the degree to which functional and structural analogies are relevant remains poorly understood. This thesis describes a multidisciplinary approach toward understanding the structure and function of TRP channel transmembrane domains by focusing on the S1-S4 transmembrane helices of the TRPV1. This focus is inspired by the voltage-sensor domain (VSD) of the S1-S4 helices of voltage-gated channels, for which a range of studies show functional and structural independence. While some TRP channels are voltage-sensitive, their S4 helix does not contain the positive string of amino acids of canonical VSDs. However, the S1-S4 helices are functionally significant as the binding site of small molecule ligands in both TRPV1 and TRPM8 (for capsaicin and menthol, respectively). The question of TRP channel transmembrane domain modularity is addressed in this thesis by expression and purification trials as well as radioligand-binding assays. It is demonstrated that the S1-S4 and S1-S6 helices of TRPV1 can be properly inserted, overexpressed, and show signs of stability upon detergent-extraction from Saccharomyces cerevisiae membranes. However the TRPV1 S1-S4 and S1-S6 helices do not show wildtype (WT)-like binding in [³H]-RTX binding assays. These results indicate that the TRPV1 transmembrane domains are likely structural but not functional domains. The S. cerevisiae expression system remained promising for the overexpression of TRP transmembrane domains as well as the production of functional, though not stable upon detergent-extraction, WT TRPV1. This WT TRPV1 was subsequently found to functionally bind both RTX, used in ligand binding assays, as well as the double-knot toxin (DkTx), targeted to the pore domain (the S5-S6 helices). An effect of DkTx on RTX binding affinity demonstrates an allosteric interaction indicative of a possible tighter packing between the two transmembrane domains than is seen in voltage-gated channels containing the canonical VSD. Computational approaches additionally allowed for the investigation of the intramembrane capsaicin binding site in the TRPV1 S1-S4 helices, crucial to the initial motivations of this study. While the literature locates the capsaicin binding site to the TRPV1 S1-S4 helices, a `binding pocket' has yet to be defined, with regards to the orientation of bound capsaicin and its access route to the site via the bilayer. Using molecular dynamics (MD) simulations the preferred location of capsaicin within the bilayer is defined, as well as the elucidiation of capsaicin flip-flop between bilayer leaflets as a key event prior to TRPV1 binding. A transient binding was also observed between a homology model of the TRPV1 S1-S4 helices and capsaicin, possibly encouraging the idea that the S1-S4 helices still contain a partial binding site, though of too low affinity to be observed in the binding experiments performed here.

血管內膜的進行性增厚是動脈粥樣硬化的重要標誌，並最終導致閉塞性血管病。血管內膜增生的一個主要因素是血管中膜的平滑肌細胞遷移至內膜層並增殖。大量研究證實，在動脈粥樣硬化的發生發展中，過量產生的活性氧簇（ROS）參與了血管壁的增厚。M型瞬時受體電位通道亞家族的成員TRPM2在血管平滑肌細胞中有表達，它能被ROS激活並對Ca²⁺通透，但其在血管平滑肌中的功能以及與心血管疾病的聯繫尚未見報道。 / 本論文著眼於探討TRPM2在鼠和人血管內膜增生中的作用。用血管外周套管法建立在體齧齒類動脈內膜增生模型。套管放置2周後，大鼠股動脈可見明顯的內膜增厚。免疫染色顯示新生內膜及其鄰近中膜區域內有大量增殖細胞核抗原陽性細胞，提示在增生的動脈中，細胞週期活動增強。動脈內膜和中膜内二氫乙錠螢光信號顯著增強，提示了ROS的過量生成。免疫染色和免疫印跡法均顯示，套管損傷導致TRPM2表達上調。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示內膜區域有大量TRPM2陽性的平滑肌細胞。與正常股動脈中膜平滑肌細胞相比，次黃嘌呤和黃嘌呤氧化酶在套管損傷的動脈來源的新生內膜平滑肌細胞中引起更大幅度的細胞內鈣離子濃度升高，而TRPM2抑制性抗體TM2E3預處理可消除這種差異。套管放置3周可引起小鼠頸動脈新生內膜形成，並伴隨著TRPM2表達上調。敲除TRPM2基因可顯著抑制內膜增生。取冠狀動脈搭橋術後殘餘的大隱靜脈，離體培養2周誘導內膜增生。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示新生內膜內含有大量TRPM2陽性的平滑肌細胞。TM2E3和另一TRPM2抑制劑2-氨乙氧基二苯酯硼酸處理均可有效降低內膜的增生。培養齧齒類主動脈平滑肌細胞，用劃痕試驗和MTT法檢測TRPM2阻斷劑和TRPM2基因敲除對過氧化氫誘導的細胞遷移和增殖的影響。結果顯示，暴露於過氧化氫48小時，細胞的遷移和增殖均明顯加快。TM2E3和2-氨乙氧基二苯酯硼酸處理有效抑制過氧化氫誘導的大鼠主動脈平滑肌細胞遷移和增殖；類似地，TRPM2基因敲除可顯著抑制過氧化氫誘導的小鼠主動脈平滑肌細胞遷移和增殖。 / 以上結果表明，血管內膜增生伴隨著TRPM2表達的上調；TRPM2參與了血管內膜增生以及血管平滑肌細胞的遷移、增殖；抑制TRPM2可能是對抗血管內膜增厚的潛在治療手段。 / A hallmark in atherosclerosis is progressive intimal thickening, which leads to occlusive vascular diseases. A causation of neointimal hyperplasia is the migration of medial smooth muscle cells (SMCs) to the intima where they proliferate. It is well recognized that excessive production of reactive oxide species (ROS) contributes to vascular wall thickening during arteriosclerotic development. TRPM2, a member of the melastatin-like transient receptor potential channel subfamily, is a Ca²⁺-permeable cation channel activated by ROS and is expressed in vascular smooth muscle cells (VSMCs). The functional properties of TRPM2 in vascular smooth muscle remain to be identified and an association between TRPM2 and cardiovascular diseases has not been reported. / In the present study, I investigated the involvement of TRPM2 in rodent and human neointimal hyperplasia. In vivo neointimal hyperplasia in rodent arteries was induced by perivascular cuff placement. After the cuff placement for 2 weeks, rat femoral arteries showed distinct intimal thickening. Immunostaining showed a great number of PCNA-positive proliferating cells in the neointima and its adjacent media region, indicating the enhanced cell cycle activity in the hyperplasic arteries. Dihydroethidium signal was markedly increased in the neointima and media of the cuffed arteries, suggesting that ROS is over-produced. Interestingly, both immunostaining and immunoblot showed that cuff-injury also led to an up-regulated expression of TRPM2. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed a large amount of TRPM2-positive SMCs in the neointimal region. Compared with the normal medial SMCs isolated from non-cuffed arteries, the neointimal SMCs from cuff-injured arteries displayed a greater [Ca²⁺] rise in response to hypoxanthine-xanthine oxidase, which was inhibited by pre-treatment with a TRPM2-specific blocking antibody TM2E3. In mouse carotid arteries, cuff placement for 3 weeks caused clear neointimal formation, accompanied by up-regulated expression of TRPM2. Trpm2 disruption dramatically reduced the neointimal growth. Human saphenous vein samples obtained during CABG surgery were organ-cultured for 2 weeks to allow growth of neointima. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed that the neointima contained numerous TRPM2-positive SMCs. Neointimal hyperplasia in the veins was effectively suppressed by in vitro treatment with TM2E3 or a chemical blocker 2-aminoethoxydiphenyl borate. Furthermore, the effect of TRPM2 blockers and Trpm2 disruption on hydrogen peroxide-induced migration and proliferation of cultured rodent aortic SMCs were evaluated by scratch wound healing assay and MTT assay, respectively. It was found that exposure to hydrogen peroxide for 48 hour substantially enhanced the migration and proliferation of rodent aortic SMCs. In rat aortic SMCs, both TM2E3 and 2-aminoethoxydiphenyl borate significantly inhibited the hydrogen peroxide-induced cell migration and proliferation. The hydrogen peroxide-induced cell migration and proliferation of SMCs was also reduced in Trpm2 knockout mice. / Taking together, these results provide strong evidences that in vivo neointimal hyperplasia is accompanied by an up-regulated expression of TRPM2 and that TRPM2 plays a key role in neointimal hyperplasia, VSMCs migration and proliferation. Blocking TRPM2 can be a potential therapeutic approach for protecting blood vessels against intimal thickening. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ru, Xiaochen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 125-151). / Abstracts also in Chinese. / Declaration of Originality --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Abbreviations and Units --- p.vii / Table of Content --- p.x / List of Figures --- p.xvi / List of Tables --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Neointimal hyperplasia --- p.1 / Chapter 1.1.1 --- Definition of neointimal hyperplasia --- p.2 / Chapter 1.1.2 --- Medical significance of coronary neointimal hyperplasia --- p.3 / Chapter 1.1.3 --- Pathogenesis of neointimal hyperplasia --- p.5 / Chapter 1.1.3.1 --- “Response to injury“ hypothesis --- p.6 / Chapter 1.1.3.2 --- Role of VSMCs --- p.7 / Chapter 1.1.3.2.1 --- VSMC phenotypic switch --- p.7 / Chapter 1.1.3.2.2 --- Ca²⁺ channel modulation in VSMCs --- p.8 / Chapter 1.1.3.2.3 --- VSMC migration --- p.9 / Chapter 1.1.3.2.4 --- VSMC proliferation --- p.10 / Chapter 1.1.3.2.5 --- Extracellular matrix production by VSMCs --- p.11 / Chapter 1.1.3.3 --- Endothelial dysfunction --- p.11 / Chapter 1.1.3.4 --- Platelet adhesion --- p.12 / Chapter 1.1.3.5 --- Inflammation --- p.13 / Chapter 1.1.4 --- Role of ROS in neointimal hyperplasia --- p.14 / Chapter 1.1.4.1 --- Types of ROS --- p.15 / Chapter 1.1.4.1.1 --- Superoxide anion --- p.16 / Chapter 1.1.4.1.2 --- Hydroxyl radical --- p.16 / Chapter 1.1.4.1.3 --- Hydrogen peroxide --- p.16 / Chapter 1.1.4.1.4 --- Nitric oxide --- p.17 / Chapter 1.1.4.2 --- Sources of ROS in vessel wall --- p.17 / Chapter 1.1.4.3 --- ROS signaling in endothelial cells --- p.19 / Chapter 1.1.4.4 --- ROS signaling in VSMCs --- p.20 / Chapter 1.1.4.5 --- ROS and atherosclerosis --- p.21 / Chapter 1.1.5 --- Current therapeutic approaches to neointimal hyperplasia --- p.23 / Chapter 1.1.5.1 --- Pharmacological approaches --- p.23 / Chapter 1.1.5.2 --- Technical Approaches --- p.25 / Chapter 1.2 --- Transient receptor potential melastatin 2 (TRPM2) channel --- p.27 / Chapter 1.2.1 --- TRP Channels --- p.27 / Chapter 1.2.2 --- TRPM2 structure and expression --- p.29 / Chapter 1.2.2.1 --- Structure --- p.29 / Chapter 1.2.2.2 --- Alternative splicing isoforms --- p.30 / Chapter 1.2.2.3 --- Expression pattern --- p.32 / Chapter 1.2.3 --- TRPM2 channel properties --- p.32 / Chapter 1.2.4 --- TRPM2 activators and inhibitors --- p.32 / Chapter 1.2.4.1 --- Activators --- p.33 / Chapter 1.2.4.1.1 --- ADPR --- p.33 / Chapter 1.2.4.1.2 --- NAD, cADPR and NAADP --- p.33 / Chapter 1.2.4.1.3 --- H₂O₂ and oxidative stress --- p.34 / Chapter 1.2.4.1.4 --- Ca²⁺ --- p.34 / Chapter 1.2.4.1.5 --- Other regulators --- p.35 / Chapter 1.2.4.2 --- Inhibitors --- p.35 / Chapter 1.2.5 --- Biological relevance of TRPM2 --- p.36 / Chapter 1.2.5.1 --- TRPM2 in insulin release --- p.36 / Chapter 1.2.5.2 --- TRPM2 in inflammation --- p.36 / Chapter 1.2.5.3 --- TRPM2 in cell death --- p.37 / Chapter 1.2.5.4 --- TRPM2-mediated lysosomal Ca²⁺ release --- p.38 / Chapter 1.2.5.5 --- TRPM2 and cardiovascular diseases --- p.39 / Chapter Chapter 2 --- Objectives of the Present Study --- p.40 / Chapter Chapter 3 --- Materials and Methods --- p.42 / Chapter 3.1 --- Materials --- p.42 / Chapter 3.1.1 --- Chemicals --- p.42 / Chapter 3.1.2 --- Media, supplements and other reagents for cell/tissue culture --- p.44 / Chapter 3.1.3 --- Antibodies --- p.45 / Chapter 3.1.4 --- Solutions --- p.46 / Chapter 3.1.4.1 --- Solutions for immunohistochemical and immunocytochemical staining --- p.46 / Chapter 3.1.4.2 --- solutions for immunoblotting --- p.47 / Chapter 3.1.4.3 --- Solutions for Genotyping --- p.49 / Chapter 3.1.4.4 --- Solutions for hematoxylin and eosin (HE) staining --- p.50 / Chapter 3.1.4.5 --- Solutions for [Ca²⁺]i measurement --- p.51 / Chapter 3.1.4.6 --- Solutions for IgG purification --- p.51 / Chapter 3.1.5 --- Animals --- p.51 / Chapter 3.1.5.1 --- Rat --- p.51 / Chapter 3.1.5.2 --- Trpm2 knockout mice --- p.52 / Chapter 3.1.5.3 --- Rabbit --- p.52 / Chapter 3.1.5.4 --- Ethics --- p.52 / Chapter 3.1.6 --- Human Tissue --- p.52 / Chapter 3.2 --- Methods --- p.54 / Chapter 3.2.1 --- Rodent models of neointimal hyperplasia --- p.54 / Chapter 3.2.1.1 --- Cuff-induced vascular injury in rat femoral artery --- p.54 / Chapter 3.2.1.2 --- Cuff-induced vascular injury in mouse carotid artery --- p.54 / Chapter 3.2.2 --- Genotyping for Trpm2 knockout mice --- p.55 / Chapter 3.2.2.1 --- Genomic DNA extraction from tail --- p.55 / Chapter 3.2.2.2 --- Polymerase Chain Reaction (PCR) --- p.55 / Chapter 3.2.2.3 --- Agarose gel electrophoresis of DNA --- p.56 / Chapter 3.2.3 --- Human saphenous vein culture and treatment --- p.56 / Chapter 3.2.4 --- Generation of anti-TRPM2 antibody, TRPM2-specific blocking antibody TM2E3 and preimmune IgG --- p.57 / Chapter 3.2.5 --- Histological analysis and immunohistochemistry --- p.58 / Chapter 3.2.6 --- Western blotting --- p.59 / Chapter 3.2.7 --- Detection of ROS production by dihydroethidium fluorescence --- p.60 / Chapter 3.2.8 --- Isolation of rodent neointimal and medial smooth muscle cells --- p.60 / Chapter 3.2.9 --- Culture of rodent aortic smooth muscle cells --- p.61 / Chapter 3.2.9.1 --- Cell culture --- p.61 / Chapter 3.2.9.2 --- Cell identification --- p.61 / Chapter 3.2.10 --- [Ca²⁺]i measurement --- p.62 / Chapter 3.2.11 --- Cell proliferation assay --- p.63 / Chapter 3.2.12 --- Cell migration assay --- p.63 / Chapter 3.2.13 --- Statistical analysis --- p.64 / Chapter Chapter 4 --- ROS over-production and TRPM2 up-regulation in cuff-induced rodent neointimal hyperplasia --- p.65 / Chapter 4.1 --- Introduction --- p.65 / Chapter 4.2 --- Materials and Methods --- p.66 / Chapter 4.2.1 --- Cuff-induced vascular injury in rat femoral artery --- p.66 / Chapter 4.2.2 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.66 / Chapter 4.2.3 --- Histological analysis and immunohistochemistry --- p.66 / Chapter 4.2.4 --- Western blotting --- p.67 / Chapter 4.2.5 --- Detection of ROS production --- p.67 / Chapter 4.2.6 --- Isolation of rat neointimal and medial smooth muscle cells --- p.68 / Chapter 4.2.7 --- [Ca²⁺]i measurement --- p.68 / Chapter 4.2.8 --- Statistical analysis --- p.68 / Chapter 4.3 --- Results --- p.69 / Chapter 4.3.1 --- Cuff-induced neointimal hyperplasia in rat femoral arteries --- p.69 / Chapter 4.3.2 --- ROS over-production in neointimal region of cuff-injured rat femoral arteries --- p.69 / Chapter 4.3.3 --- TRPM2 up-regulation in neointimal region of cuff-injured rat femoral arteries --- p.69 / Chapter 4.3.4 --- Enhanced [Ca²⁺]i response to HX-XO in rat neointimal smooth muscle cells --- p.70 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- TRPM2 contributes to human and rodent neointimal hyperplasia --- p.86 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Materials and Methods --- p.87 / Chapter 5.2.1 --- Cuff-induced vascular injury in mouse carotid artery --- p.87 / Chapter 5.2.2 --- Genotyping for Trpm2 knockout mice --- p.87 / Chapter 5.2.3 --- Organ culture of human saphenous vein --- p.87 / Chapter 5.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.88 / Chapter 5.2.5 --- Histological analysis and immunohistochemistry --- p.88 / Chapter 5.2.6 --- Western blotting --- p.88 / Chapter 5.2.7 --- Isolation of mouse neointimal and medial smooth muscle cells --- p.89 / Chapter 5.2.8 --- [Ca²⁺]i measurement --- p.89 / Chapter 5.2.9 --- Statistical analysis --- p.90 / Chapter 5.3 --- Results --- p.90 / Chapter 5.3.1 --- Cuff-induced neointimal hyperplasia was reduced in Trpm2 knockout mice --- p.90 / Chapter 5.3.2 --- [Ca²⁺]i response to HX-XO in mouse neointimal smooth muscle cells --- p.90 / Chapter 5.3.3 --- Inhibiting TRPM2 reduced the neointimal hyperplasia in in vitro cultured human saphenous vein --- p.91 / Chapter 5.4 --- Discussion --- p.99 / Chapter Chapter 6 --- Role of TRPM2 in H₂O₂-stimulated migration and proliferation of vascular smooth muscle cells --- p.103 / Chapter 6.1 --- Introduction --- p.103 / Chapter 6.2 --- Materials and Methods --- p.104 / Chapter 6.2.1 --- Culture of rodent aortic smooth muscle cells --- p.104 / Chapter 6.2.2 --- Immunocytochemistry --- p.104 / Chapter 6.2.3 --- Genotyping for Trpm2 knockout mice --- p.104 / Chapter 6.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.104 / Chapter 6.2.5 --- [Ca²⁺]i measurement --- p.105 / Chapter 6.2.6 --- Cell proliferation assay --- p.105 / Chapter 6.2.7 --- Western blotting --- p.105 / Chapter 6.2.8 --- Cell migration assay --- p.106 / Chapter 6.2.9 --- Statistical analysis --- p.106 / Chapter 6.3 --- Results --- p.106 / Chapter 6.3.1 --- H₂O₂-induced [Ca²⁺]i rises in rodent aortic smooth muscle cells --- p.106 / Chapter 6.3.2 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell proliferation --- p.107 / Chapter 6.3.3 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell migration --- p.108 / Chapter 6.4 --- Discussion --- p.118 / Chapter Chapter 7 --- General Conclusion and Future Work --- p.121 / Chapter 7.1 --- Concluding remarks --- p.121 / Chapter 7.2 --- Future work --- p.123 / Chapter 7.2.1 --- Specific downstream signaling pathway of TRPM2 that mediates ROS-induced VSMC proliferation and migration --- p.123 / Chapter 7.2.2 --- Involvement of TRPM2 in leukocyte infiltration and inflammation in vascular wall --- p.124 / References --- p.125 / List of Publications --- p.152

壓力感受器在調節血壓的壓力感受性反射中的作用已是眾所周知。兩個動脈壓力感受器，分別為主動脈壓力感受器和頸動脈壓力感受器。它們作為重要的感應器以檢測主要動脈血壓，並和孤束核溝通，從而調節血壓。然而，壓力感受器的機械力敏感元件的分子身份仍是奧秘。因為機械敏感的陽離子通道受機械力刺激時會增加的神經元活動, 所以機械敏感的陽離子通道是合適的候選人。 / 在本研究中，通過使用膜片鉗和動作電位的測量，瞬时受体电位通道C5(TRPC5)被確定在主動脈壓力感受器的機械傳感器中。透過在壓力感受器神經元的鈣測量實驗，證實TRPC5參與由拉伸引起的鈣離子([Ca²⁺]i)上升。TRPC5基因敲除小鼠出現壓力感受器功能受損, 表明了TRPC5在血壓控制的重要性。 / 比較主動脈壓力感受器或頸動脈壓力感受器的不同敏感度現時存有不少爭論。在本研究中，我發現主動脈壓力感受器比頸動脈壓力感受器對於壓力變化更加敏感。此外，我還發現了主動脈壓力感受器神經元比頸動脈壓力感受器神經元有一個相對較高的瞬时受体电位通道V4(TRPV4)表達。鈣測量研究表明TRPV4通道在主動脈壓力感受器神經元的靈敏度可能發揮著重要作用。 / Baroreceptors have been well known for its role in the baroreflex regulation of blood pressure. Two arterial baroreceptors, aortic and carotid baroreceptors, serve as the important sensors to detect blood pressure in main arteries, and they communicate with the solitary nucleus tract for blood pressure regulation. However, the molecular identity of the mechano-sensitive components in the baroreceptors is still mysteries. Mechano-sensitive cation channels are the fascinating candidates as they increase neuronal activities when stimulated by stretch. In the present study, with the use of patch clamp and action potential measurement, TRPC5 channels were identified to be the mechanical sensor in the aortic baroreceptor. Calcium measurement studies demonstrated that TRPC5 was involved in the stretch-induced [Ca2+]i rise in baroreceptor neurons. The importance of TRPC5 in blood pressure control was also studied in TRPC5 knockout mice, which displayed an impaired baroreceptor function. / There have been controversies as to whether aortic baroreceptors or carotid baroreceptors are more sensitive to the change in blood pressure. In the present study, aortic baroreceptor was found to be more sensitive to the pressure change than the carotid baroreceptor. Furthermore, I also found a relative higher expression of TRPV4, a mechanosensitive channel, in the aortic baroreceptor neurons than in the carotid baroreceptor neurons. Moreover, calcium measurement studies showed that TRPV4 channels should play an important role in governing the differential pressure sensitivity in these two types of baroreceptor neurons. / Taken together, the present study provided novel information on the role of TRPC5 and TRPV4 in baroreceptor mechanosensing. In future, it will be of interest to explore whether TRPC5 and/or TRPV4 dysfunction could contribute to human diseases that are related to blood pressure control. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lau, On Chai Eva. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 133-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Declaration --- p.i / Abstract of the thesis entitled --- p.ii / Acknowledgement --- p.vii / Abbreviation --- p.ix / Table of content --- p.xii / List of figures --- p.xv / List of table --- p.xvii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Baroreceptors --- p.1 / Chapter 1.1.2 --- Arterial baroreceptors --- p.2 / Chapter 1.1.2.1 --- Functions of arterial baroreceptors --- p.4 / Chapter 1.1.2.2 --- Sensitivity of the arterial baroreceptors --- p.6 / Chapter 1.1.3 --- Other baroreceptors --- p.8 / Chapter 1.1.4 --- The molecular identity of the mechanosensors in baroreceptor neurons --- p.9 / Chapter 1.2 --- Transient receptor potential ion channels (TRP channels) --- p.10 / Chapter 1.2.1 --- TRP channels superfamily --- p.10 / Chapter 1.2.2 --- Multimerization of TRP channels --- p.12 / Chapter 1.2.3 --- Physiological functions --- p.14 / Chapter 1.2.4 --- Mechanosensitive TRP channels --- p.16 / Chapter 1.2.5 --- Canonical transient receptor potential 5 (TRPC5) channels --- p.17 / Chapter 1.2.6 --- Vanilloid transient receptor potential 4 (TRPV4) channels Figures --- p.20 / Chapter Chapter 2: --- Objectives --- p.34 / Chapter Chapter 3: --- Materials and Methods --- p.35 / Chapter 3.1 --- Materials --- p.35 / Chapter 3.1.1 --- Chemicals and reagents --- p.35 / Chapter 3.1.2 --- Solutions --- p.36 / Chapter 3.1.2.1 --- Solutions for calcium imaging --- p.36 / Chapter 3.1.2.2 --- Solutions for electrophysiology study --- p.38 / Chapter 3.1.2.3 --- Solutions for agarose gel electrophoresis --- p.41 / Chapter 3.1.3 --- Primers for RT-PCR --- p.42 / Chapter 3.1.4 --- Animals --- p.43 / Chapter 3.2 --- Methods --- p.43 / Chapter 3.2.1 --- Total RNA isolation and RT-PCR --- p.43 / Chapter 3.2.2 --- Immunohistochemistry --- p.44 / Chapter 3.2.3 --- Neuron labeling by DiI --- p.45 / Chapter 3.2.4 --- Neuron culture --- p.46 / Chapter 3.2.5 --- [Ca²⁺]i measurement --- p.47 / Chapter 3.2.6 --- Electrophysiology --- p.48 / Chapter 3.2.7 --- Evaluation of baroreflex response --- p.49 / Chapter 3.2.8 --- Telemetric measurement of blood pressure --- p.50 / Chapter 3.2.9 --- Statistical analysis --- p.51 / Figures --- p.52 / Chapter Chapter 4: --- Functional role of TRPC5 channels in aortic baroreceptor --- p.56 / Chapter 4.1 --- Introduction --- p.56 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Animals --- p.59 / Chapter 4.2.2 --- Immunohistochemistry --- p.59 / Chapter 4.2.3 --- Neuron labeling by DiI --- p.61 / Chapter 4.2.4 --- Neuron culture --- p.62 / Chapter 4.2.5 --- [Ca²⁺]i measurement --- p.63 / Chapter 4.2.6 --- Electrophysiology --- p.63 / Chapter 4.2.7 --- Evaluation of baroreflex response --- p.64 / Chapter 4.2.8 --- Telemetric measurement of blood pressure --- p.66 / Chapter 4.2.9 --- Statistical analysis --- p.67 / Chapter 4.3 --- Results --- p.67 / Chapter 4.3.1 --- Endogenous expression of TRPC5 channels in aortic baroreceptor neurons --- p.67 / Chapter 4.3.2 --- Characterization on the pressure-sensitive component in aortic baroreceptors --- p.68 / Chapter 4.3.3 --- Involvement of TRPC5 in [Ca²⁺]i response in aortic baroreceptor neurons --- p.69 / Chapter 4.3.4 --- Participation of TRPC5 in pressure-induced action potential firing in cultured aortic baroreceptor neurons --- p.70 / Chapter 4.3.5 --- Role of TRPC5 in baroreceptor sensory nerve activity and baroreflex regulation --- p.71 / Chapter 4.3.6 --- Importance of TRPC5 in baroreceptor function --- p.72 / Chapter 4.4 --- Discussions --- p.74 / Figures --- p.79 / Table --- p.98 / Chapter Chapter --- 5: TRPV4 channels and baroreceptor sensitivity --- p.99 / Chapter 5.1 --- Introduction --- p.99 / Chapter 5.2 --- Materials and Methods --- p.101 / Chapter 5.2.1 --- Animals --- p.101 / Chapter 5.2.2 --- Neuron labeling by DiI --- p.101 / Chapter 5.2.3 --- Neuron culture --- p.102 / Chapter 5.2.4 --- Electrophysiology --- p.103 / Chapter 5.2.5 --- Immunohistochemistry --- p.104 / Chapter 5.2.6 --- [Ca²⁺]i measurement --- p.105 / Chapter 5.2.7 --- Statistical analysis --- p.105 / Chapter 5.3 --- Results --- p.106 / Chapter 5.3.1 --- Properties of the aortic and carotid baroreceptor neurons --- p.106 / Chapter 5.3.2 --- Stretch sensitivity of aortic and carotid baroreceptor neurons --- p.108 / Chapter 5.3.3 --- mRNA expression of mechanosensitive TRP channels in aortic and carotid baroreceptor neurons --- p.109 / Chapter 5.3.4 --- Protein expression of TRPV4 channels in aortic and carotid baroreceptor neurons --- p.109 / Chapter 5.3.5 --- Involvement of TRPV4 in stretch-induced [Ca²⁺]i response in baroreceptor neurons --- p.110 / Chapter 5.4 --- Discussions --- p.111 / Figures --- p.116 / Chapter Chapter 6: --- General conclusions and future directions --- p.124 / Figures --- p.128 / References --- p.133

Transient receptor potential (TRP) channels comprise a superfamily of cation-selective ion channels that are largely calcium (Ca2+) permeable and that play diverse physiological roles ranging from nociception in primary afferent neurons to the absorption of dietary Ca2+. The 28 mammalian TRP channels are categorized into 6 subfamilies. The vanilloid subfamily is named for its founding member, TRPV1, the capsaicin receptor, and has 6 members. TRPV1-4 are all heat sensitive ion channels whereas TRPV5 and TRPV6 are involved in renal Ca2+ reabsorption and Ca2+ absorption in the intestine, respectively. In our structural studies, we have focused on TRPV3 and TRPV6. TRPV6 is a highly Ca2+ selective TRP channel (PCa/PNa ~ 130) that functions in active Ca2+ absorption in the intestine. Its expression is upregulated by vitamin D and is, on the molecular level, regulated by PIP2 and calmodulin (CaM). Previously, the structure of TRPV6 was solved using X-ray crystallography. Using the crystal structure, a negatively charged extracellular vestibule was identified and anomalous diffraction was used to identify ion binding sites in the pore. Also, at the top of the selectivity filter, four aspartates were identified that coordinate Ca2+ entering the pore and confer to TRPV6 its selectivity for Ca2+. However, only the structure of the rat orthologue was solved and only in the closed, apo state. We used cryo-electron microscopy (cryo-EM) to solve structures of the human orthologue of TRPV6 in the open and closed (we used the mutation R470E to close the channel) states. The closed-to-open TRPV6 transition is accompanied by the formation of short π-helices in the middle of the pore-lining S6 helices, which in turn results in their turning and a different set of residues facing the pore. Additionally, the formation of the π-helices results in kinking of the S6 helices, which further widens the pore. TRPV6 is constitutively active when expressed heterologously. In other words, the addition of external stimuli is not necessary for the activation of the channel. Therefore, its activity needs to be regulated to prevent toxic Ca2+ overload. One mechanism by which this occurs is through its regulation by CaM. CaM has been shown to bind TRPV6 and regulate its function, however, the way it binds to and regulates TRPV6 remained unknown. To uncover this mechanism, we solved the structure of TRPV6 bound to CaM. We found that CaM binds TRPV6 in a 1:1 stoichiometric ratio and that CaM directly blocks the TRPV6 pore by inserting a positively charged lysine into a tera-tryptophan cage at the bottom of the pore. As a result, the channel adopts an inactivated conformation; although the pore-lining S6 helices still contain local π-helices, they are pulled closer together, narrowing the pore and further blocking it with hydrophobic side chains. We have also conducted studies of TRPV3. Unlike TRPV6, TRPV3 is a heat-activated vanilloid TRP channel. TRPV3 is expressed highly in keratinocytes where it has been implicated in wound healing and maintenance of the skin barrier, and in the regulation of hair growth. We solved the structure of apo TRPV3 in a closed state, and the structure of a TRPV3 mutant bound to 2-APB in an open state. Like TRPV6, the opening of TRPV3 is accompanied by the formation of local π-helices in the middle of the pore-lining S6 helices. The formation of the π-helices results in the lining of the ion permeation pathway with a different set of residues, resulting in a largely negatively charged pathway. Unlike TRPV6, TRPV3 is only slightly selective for Ca2+ and correspondingly, during gating state transitions, rearrangements were not only observed only in its pore-lining helices, but also in the cytosolic domain and the selectivity filter. Based on a comparison of our structures, we proposed a model of TRPV3 regulation by 2-APB. Together, our studies provide insight into the regulatory and gating mechanisms of the vanilloid subtype TRP channels and can provide the foundation for future studies.

Thesis (Ph.D.)--University of Central Florida, 2008. / Adviser: Sic L. Chan. Includes bibliographical references (p. 74-84).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 338-360).

We have studied the modulation of gating properties of the Ca2+-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4αPDD transiently activated a current through TRPV4 in the presence of extracellular Ca2+. Increasing the concentration of extracellular Ca2+ ([Ca2+]e) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca2+]e. It was also much slower in the presence of [Ca2+]e in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797 , were constitutively open. In conclusion, gating of the 4αPDD-activated TRPV4 channel depends on both extra- and intracellular Ca2+, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.