Wong, Ching On. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 150-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Mode of access: World Wide Web. / Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 102-113). / Electronic reproduction. / Also available by subscription via World Wide Web / xvi, 113, pp leaves, bound ill. (some col.) 29 cm

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xviii, 178 p.; also includes graphics. Includes bibliographical references (p. 163-178). Available online via OhioLINK's ETD Center

Thesis (Ph.D.)--University of California, San Francisco, 2005. / Includes bibliographical references. Also available online.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

The caudal nucleus tractus solitarius (cNTS) is the key recipient of the primary afferents from visceral sensory neurons and also an important site that processes and integrates gastrointestinal, cardiovascular and respiratory functions. Glutamate and gamma-aminobutyric acid are the major neurotransmitters within the NTS, but studies have suggested that nicotinic acetylcholine receptors (nAChRs) and transient receptor potential (TRP) channels can modulate excitatory/inhibitory neurotransmission. I have designed studies to understand the role of nAChRs and TRP channels in the modulation of neurotransmission in the cNTS. In the first aim, experiments were designed to test the hypothesis that the cNTS contains function-specific subsets of neurons whose responsiveness to nicotine correlates with the target of their axonal projections. cNTS neurons send axonal projections to brain regions such as parabrachial nucleus (PBN), hypothalamic paraventricular nucleus (PVN), nucleus ambiguous (NA), dorsal motor nucleus of the vagus (DMV) and the caudal ventrolateral medulla (CVLM) and are involved in integrating autonomic and neuroendocrine functions. Presynaptic/postsynaptic modulation by nAChRs differ in the axonal projections of cNTS neurons, studying of which would provide better understanding of this complex integration. In vivo fluorescent tracing combined with in vitro slice patch-clamp electrophysiological recordings from anatomically identified caudal NTS neurons were used to study the expression and function of nAChRs (mainly á3â4 containing nAChRs) in the cNTS. Results from these studies demonstrate that presynaptic and postsynaptic responsiveness of caudal NTS neurons to nicotine correlates with the areas the neurons project to in the following order of prevalence: DMV>PVN>NA>CVLM>PBN (for presynaptic responses) and DMV>CVLM>PBN>NA>PVN (for postsynaptic responses). In the second aim, experiments were designed to test the hypothesis that nociceptive TRP channels TRPV1 (vanilloid) and TRPA1 (ankyrin) modulate synaptic transmission in the NTS. As a result of this modulation, the efferent functions that control autonomic and visceral functions will be regulated and account for the changes in autonomic neuropathy as patients with diabetes develop significant alterations in blood pressure and heart rate as well as silent myocardial ischemia as a result of blunted pain carrying ability. Results obtained from these studies demonstrated that TRPV1 and TRPA1 mRNA were detected in the dorsal root ganglion (DRG), but not in the NTS. Immunofluorescence studies revealed that TRPV1 and TRPA1 were expressed in the solitary tract central sensory terminals inputs to NTS but not in NTS neurons. This suggests that TRPV1 and TRPA1 are expressed only in solitary tract. Administration of capsaicin (TRPV1 agonist) and allyl isothiocyanate (AITC, TRPA1 agonist) both increased the frequency of s/mEPSCs without affecting spontaneous and miniature inhibitory postsynaptic currents (s/mIPSCs). Next, the modulation of TRPV1- and TRPA1-induced responses by utilizing a PKC activator (PDBu) was examined. Incubation of slices with PDBu synergistically increased the mEPSC frequency following capsaicin application suggesting an increased receptor affinity; however following application of AITC there was no significant change, suggesting that activation by covalent modification does not enhance binding affinity. Finally, the specificity of TRPV1 and TRPA1 effect on synaptic transmission by ablating TRPV1 and TRPA1were tested. There was no modulation of synaptic transmission in these animals, further confirming that capsaicin- and AITC-mediated modulation of synaptic transmission are specifically mediated by TRPV1 and TRPA1, respectively. Furthermore, animals with painful diabetic peripheral neuropthy exhibited enhanced synaptic activity at the NTS, suggesting a role in nociception and other visceral functions. In summary, nAChRs, TRPV1 and TRPA1 are expressed in the NTS and activation of which modulate excitatory synaptic transmission. The results obtained from these studies and their interpretation may provide a better understanding of the central mechanism of modulation on efferent functions from NTS that regulate cardiovascular, respiratory and gastrointestinal functions.

Calcium plays an essential role in the physiology and biochemistry of many biological functions, including excitation-contraction coupling, neuronal signaling, and fertilization. In mammals, the calcium content in various tissues, organs, and cell types is tightly regulated to maintain homeostasis. A chief process controlling calcium levels is absorption of the ion from the lumen by epithelial cells that line organs including the intestines and kidney. Calcium entry at the apical membrane constitutes the first step of epithelial calcium absorption. Two highly calcium-selective transient receptor potential vanilloid (TRPV) channels, TRPV5 and TRPV6, are the pore-forming subunits responsible for epithelial calcium entry in kidney and intestine, respectively. Genetic knockout of TRPV5 or TRPV6 in animals leads to phenotypes related to defective calcium homeostasis, including lowered serum calcium levels, decreased calcium absorption, reduced bone density, impaired sperm motility, and decreased maternal-fetal calcium transfer. In humans, aberrant TRPV5/6 expression is associated with preeclampsia and calcium nephrolithiasis (kidney stones). Additionally, TRPV6 expression level is upregulated in carcinomas of prostate, colon, breast, thyroid, and ovary, suggesting a role for TRPV6 in cancer survival. A detailed understanding of epithelial calcium entry is hindered by a lack of high-resolution structural information on intact channels. This dissertation presents structural analyses of the epithelial calcium channel TRPV6. We applied modern membrane protein screening and expression techniques, including fluorescence-detection size exclusion chromatography (FSEC) and baculovirus mediated mammalian cell transduction (BacMam), to identify optimal TRPV6 constructs and purification schemes for crystallization. Using a surface mutagenesis approach guided by lower-resolution structural solutions, we engineered a rat TRPV6 mutant (TRPV6cryst) that permitted solving a 3.25 Å resolution crystal structure. We used fluorescent calcium indicator assays to show that TRPV6cryst retains the permeation and ionic block properties of the wild type channel. The tetrameric structure of TRPV6cryst reveals a transmembrane domain architecture similar to voltage gated ion channels, with the ion conducting pore coincident with the overall four-fold symmetry axis. A ring of aspartate (D541) residues, shown in previous studies as a critical determinant of calcium selectivity, forms a narrow constriction at the extracellular pore entrance, or selectivity filter. Methionine (M577) side chains in the lower portion of the channel pore plug the conduction pathway and define the closed state of the channel. The ankyrin repeat domain, linker domain, N-terminal helix, and C-terminal hook form an intracellular skirt surrounding a cavity that lies beneath the pore axis. Close interactions between these domains, in large part mediated by the N-terminal helix, suggest that they are involved in allosteric modulation or concerted movements associated with channel activation. To shed light on the structural bases of permeation and ionic block, we cocrystallized TRPV6cryst with the permeant cations Ca²⁺ and Ba²⁺, and the channel blocker Gd³⁺. We identified binding sites for these cations by exploiting their anomalous scattering properties. On the basis of the cation-binding sites, we propose a permeation mechanism in which cations are recruited toward the pore by electronegative side chains in the extracellular vestibule, followed by sequential binding at least three binding sites along the central pore axis. Ca²⁺ selectivity is apparently achieved by high-affinity binding to the ring of D541 side chains in the selectivity filter. Gd³⁺ blocks permeation by similarly binding to the D541 ring and outcompeting ions of lesser charge. The results described in this dissertation provide a structural framework to further study mechanisms of epithelial calcium entry in health and disease.

The etiology of bipolar disorder (BD) is multidimensional and thought to involve several factors that increase neuronal oxidative stress and disrupt intracellular calcium homeostasis. As calcium-permeable canonical transient receptor potential channels (TRPC) have been linked to bipolar pathophysiology, I sought to determine whether oxidative stress affects TRPC3/TRPC5/TRPC6 expression and/or function. Chronic (4-day) but not acute (24-hour) rotenone-induced oxidative stress dose-dependently reduced TRPC5 and TRPC6 protein levels in primary rat cortical neurons. A decrease in TRPC5 mRNA levels was only found following acute but not chronic rotenone whereas TRPC6 mRNA levels did not change significantly with either treatment. Reduced TRPC3 function was seen after chronic stress when stimulated by TRPC3/6 activator, 1-oleoyl-2-acetyl-sn-glycerol. Lithium pre-treatment attenuated the rotenone-induced reduction in TRPC3 but not TRPC6 protein levels. These results suggest TRPC subtypes are differentially regulated by oxidative stress and support a potential mechanistic link between oxidative stress and calcium dyshomeostasis in BD.

The etiology of bipolar disorder (BD) is multidimensional and thought to involve several factors that increase neuronal oxidative stress and disrupt intracellular calcium homeostasis. As calcium-permeable canonical transient receptor potential channels (TRPC) have been linked to bipolar pathophysiology, I sought to determine whether oxidative stress affects TRPC3/TRPC5/TRPC6 expression and/or function. Chronic (4-day) but not acute (24-hour) rotenone-induced oxidative stress dose-dependently reduced TRPC5 and TRPC6 protein levels in primary rat cortical neurons. A decrease in TRPC5 mRNA levels was only found following acute but not chronic rotenone whereas TRPC6 mRNA levels did not change significantly with either treatment. Reduced TRPC3 function was seen after chronic stress when stimulated by TRPC3/6 activator, 1-oleoyl-2-acetyl-sn-glycerol. Lithium pre-treatment attenuated the rotenone-induced reduction in TRPC3 but not TRPC6 protein levels. These results suggest TRPC subtypes are differentially regulated by oxidative stress and support a potential mechanistic link between oxidative stress and calcium dyshomeostasis in BD.

My PhD project investigated the regulation of human cardiac transient outward potassium current (Ito) by protein tyrosine kinases (PTKs) and the functional expression of transient receptor potential (TRP) channels in human atrial myocytes to make an advanced understanding of human cardiac electrophysiology and pathophysiology. The modulation of human cardiac Itoby PTKs was studied in human atrial myocytes and HEK 293 cells expressing hKv4.3 (coding human cardiac Ito). We found that the broad-spectrum PTK inhibitor genistein, the selective EGFR kinase inhibitor AG556, and the Src-family kinases inhibitor PP2 inhibited human atrial Itoand the inhibitory effect was countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Similar results were observed in hKv4.3-HEK cells. Interestingly, tyrosine phosphorylation of hKv4.3channels was reduced by genistein, AG556, and PP2,and the reduction was antagonized by orthovanadate. The mutant Y136F of hKv4.3 lost the inhibitory response to AG556, whileY108F lost the response to PP2.The double mutant Y108F-Y136F hKv4.3 failed to respond to both AG556 and PP2, and exhibited a dramatic reduction of tyrosine phosphorylation. These results indicate that native cardiac Itois regulated by both EGFR and Src family kinases. In the second part, we studied whether TRPC channels would mediate the nonselective cation current described previously in human atrial myocytes. It was found that TRPC1 channel activator thapsigargin activated the current, and the effect was suppressed by La3+or prevented by intracellular anti-TRPC1 antibody. Endothelin-1 and angiotensin II stimulated the current, andthe effect was inhibited by La3+and/or 2-APB. RT-PCR and Western blot analysis revealed that in addition to the TRPC1 channels mediating the nonselective cation current, the components of store-operated Ca2+channels (SOCs), STIM1 and Orai1 were abundantly expressed in human atria. The interaction of TRPC1, STIM1, and Orai1 was confirmed by co-immunoprecipitation. Interestingly, we found that protein expression of TRPC1 and STIM1, but not Orai1, was up-regulated in human atria with atrial fibrillation. The third part of the project determined whether TRPM7 channels were expressed in human atrial myocytes, since this channel was reported in human atrial fibroblasts, conferring atrial fibrosis in human atria with atrial fibrillation. We found a TRPM7 -like current which was potentiated by acidic pH, and inhibited by La3+and 2-APB, and a Ca2+-activated TRPM4 current. RT-PCR and Western blot analysis confirmed the expression of TRPM7 and TRPM4 channels in human atria. Moreover, we found TRPM7 protein, but not TRPM4 protein was significantly up-regulated in human atria with atrial fibrillation, suggesting the potential participation of TRPM7 channels in atrial remodeling of human atria with atrial fibrillation. Collectively, this PhD thesis project has demonstrated for the first time that human cardiac Itois modulated by EGFR kinase and Src kinases via phosphorylating Y136and Y108, respectively. TRPC1 channels mediate the nonselective cation current and SOCs.TRPM7 channels are expressed in human atrial myocytes. The up-regulation of TRPC1, STIM1, and TRPM7 channels in human atria with atrial fibrillation suggest that they are likely involved in atrial electrical and/or structure remodeling in patients with atrial fibrillation. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy