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.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20310号 / 薬科博第79号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 金子 周司, 教授 竹島 浩, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

L'oxaliplatine, largement utilisé dans le traitement du cancer colorectal, se singularise par la survenue très précoce, dés le début du protocole de chimiothérapie de troubles douloureux associés à la perception du froid (hypersensibilité au froid). De nombreux traitements ont été testés pour ces symptômes sans grand succès, c'est pourquoi le développement de nouveaux analgésiques est nécessaire. Le but de ce travail est de développer un modèle souris reproduisant cette hypersensibilité au froid et d'en rechercher les mécanismes physiopathologiques. Comme chez les patients, l'administration aigüe d'oxaliplatine entraîne une amplification importante de la perception du froid chez la souris. Nous montrons que ces symptômes sont médiés par les nocicepteurs exprimant le thermorécepteur TRPM8. Sur le plan du mécanisme physiopathologique, l'oxaliplatine favorise l'excitabilité de ces nocicepteurs en diminuant de manière drastique l'expression des plusieurs canaux potassiques (TREK1 et TRAAK en particulier), et en augmentant l'expression de canaux proexcitateurs tels que les canaux cationiques activés par l'hyperpolarisation (HCN1 notamment). Ces constatations sont confortées au niveau comportemental par l'analyse de la lignée de souris double KO pour TREK1 et TRAAK, et par l'utilisation de l'Ivabradine, un inhibiteur pharmacologique spécifique des canaux HCNs. L'ivabradine, déjà utilisé en clinique pour le traitement de l'insuffisance cardiaque, entraîne une disparition de l'hypersensibilité au froid induite par l'oxaliplatine. Collectivement, ces résultats suggèrent que l'oxaliplatine exacerbe la perception du froid en remodelant le patron d'expression de plusieurs canaux ioniques qui coordonnent la réponse au froid. L'ivabradine peut ainsi représenter un traitement sur mesure pour les neuropathies induites par l'oxaliplatine. / The hallmark of oxaliplatin-induced neuropathy is a hypersensibility to cold that develops in nearly all patients ultimately leading to cessation of this chemotherapy treatment. To date, classical pain management strategies have failed to alleviate these painful symptoms, and hence there is a need for developing new and efficient analgesics. Here we report that, as in patients, oxaliplatin mediates a clear development of exaggerated perception of cold temperatures in mice. These symptoms are mediated by nociceptors expressing the thermoreceptor TRPM8. Mechanistically, we find that oxaliplatin promotes excitability in nociceptors drastically lowering the expression of distinct potassium channels (TREK1, TRAAK) that act as excitability brakes for cold perception, and by increasing the expression of pro-excitatory channels such as the hyperpolarisation-activated channels (HCNs). These findings are corroborated by the analysis of the TREK1-TRAAK null mice, and by the use a specific HCN channel inhibitor abolishing the oxaliplatin-induced cold hypersensibility. Collectively, these results suggest that oxaliplatin exacerbates cold perception in cold sensing neurons by transcriptionally remodeling a combination of ionic conductances that together shape the final response to cold. A direct promising clinical consequence of these findings for patients would be that the HCN inhibitor ivabradine could represent a tailored treatment for oxaliplatin-induced neuropathy.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cinnamaldehyde, a natural compound frequently present in cosmetic formulations, induces skin irritation when topically applied, but the mechanism by which cinnamaldehyde produces such skin reactions is unclear. Here, we showed that cinnamaldehyde induced ear edema in mice (1-6 μg/ear) with a maximum effect with 4 μg/ear (Emax of 0.18 ± 0.02 mm and an ED50 value of 2.0 (1.1- 3.4 μg/ear). Cinnamaldehyde can induce leukocyte infiltration detected by an increase in MPO activity and confirmed by histological analyses. The edema and cellular infiltration evoked by 4 μg/ear of cinnamaldehyde was prevented through topical application of ruthenium red, a non selective TRP antagonist or by camphor and HC030031, two TRPA1 receptor antagonists. In contrast, the edema and the leukocyte infiltration was unaffected by the TRPV1 receptor antagonist SB366791. Cinnamaldehydeinduced edema but not cellular infiltration was also prevented though topical application of the tachykinin NK1 antagonist aprepitant, indicating a neuropeptides release phenomenon in this process. Also, we observed that repeated topical applications of cinnamaldehyde (4 μg/ear) did not induced sensitization/desensitization alterations. Interestingly, the TRPV1 antagonist, capsaicin, repeated treatment abrogated its edematogenic response, confirming the desensitization process and decrease partially the cinnamaldehyde induced edema, suggesting the involvement of capsaicin-sensitive fibers and additional targets in cinnamaldehyde response. The present results demonstrated that cinnamaldehyde induces mouse skin inflammation through a mechanism involved the TRPA1 receptor activation and subsequent leukocyte infiltration. In addition, evidence supports the assumption that the tachykinin NK1 receptor is involved in these inflammatory responses. / O cinamaldeído é um composto natural frequentemente encontrado em formulações cosméticas, capaz de induzir irritação na pele quando aplicado topicamente, porém o mecanismo pelo qual o cinamaldeído produz estas reações ainda é desconhecido. Neste trabalho demonstramos que o cinamaldeído foi capaz de induzir edema de orelha em camundongos (1-6 μg/orelha) com um efeito máximo obtido com a dose de 4 μg/orelha (Emax de 0,18 ± 0,02 mm e um DE50 de 2,0 (1,1- 3,4) μg/orelha). O cinamaldeído foi capaz ainda de induzir infiltração leucocitária detectada por um aumento na atividade da MPO e confirmada por análise histológica. O edema e a infiltração leucocitária iniciados após aplicação tópica de 4 μg/orelha de cinamaldeído foi prevenido pela aplicação tópica de vermelho de rutênio, um antagonista TRP não seletivo, e por cânfora e HC030031, dois antagonistas seletivos TRPA1. Por outro lado, a aplicação de SB366791, um antagonista seletivo TRPV1, não alterou o edema nem a infiltração leucocitária. Ainda, o edema induzido pelo cinamaldeído foi prevenido pela aplicação tópica de aprepitant, um antagonista seletivo do receptor NK1 para taquicininas, sugerindo que a liberação de neuropeptídeos esteja envolvida neste processo. Também foi observado que a aplicação tópica repetida de cinamaldeído 4 μg/orelha não foi capaz de induzir processos de ensibilização/dessensibilização. No entanto, o tratamento repetidocom o antagonista TRPV1, capsaicina, aboliu o edema induzido pela própria capsaicina, confirmando a ocorrência de dessensibilização, e diminuiu parcialmente o edema induzido pelo cinamaldeído sugerindo o envolvimento de fibras sensíveis a capsaicina, além de outras vias, neste processo. Os resultados demonstram que o cinamaldeído induz um processo inflamatório na pele através de um mecanismo que envolve a ativação do receptor TRPA1 e consequente infiltração leucocitária.

Pour mieux comprendre les mécanismes impliqués dans la genèse et la régulation de l'activité rythmique cardiaque, une bonne connaissance des gènes impliqués dans ces mécanismes et de leur fonction est nécessaire.J'ai mis au point les conditions expérimentales d'un crible génétique et identifié deux gènes régulateur du rythme cardiaque : ork1, code pour un canal potassique à deux pores, pur régulateur du rythme cardiaque ; painless, code pour un canal TRPA, régulateur du rythme cardiaque et acteur essentiel de la réponse cardiaque au stress mécanique. Du fait de l'importance de la régulation de l'activité cardiaque par le stress acide, le rôle de NDAE, échangeur Cl/HCO3, a été recherché. Il est requis pour une meilleure récupération après un stress acide et couplé à l'échangeur Na/Ca.Ces travaux ont permis de valider le modèle Drosophile pour identifier de nouveaux gènes et pour étudier le rôle des gènes encore mal connu dans l'activité cardiaque. / A good knowledge of genes implicated in genesis of cardiac activity and in its regulation is crucial for a better understanding of arrhythmia.In this study, I have developed experimental conditions to perform a genetic screen and identified two gens implicated in cardiac activity : ork1, encoding a two-pore potassium channel is a pure regulator of cardiac rhythm ; painless, encoding a TRPA channel, regulates cardiac frequency and mediates the cardiac response to mechanical stress. We know that cardiac activity is particulary sensitive to acidic stress and the study of NDAE, the unique Drosophila Cl/HCO3 exchanger, has shown that NDAE is required for cardiac activity recovery after acidic stress and displayed the genetic link with the Na/Ca exchanger.This work validates Drosophila as a good system to found new genes implicates in cardiac activity, particularly in genetic screen, and to precise the role of genes still unknown in cardiac activity.

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.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / Los canales de la superfamilia TRP son canales catiónicos no selectivos que permean principalmente Ca2+ y Na+, y comparten una arquitectura molecular general. Las subunidades de un canal TRP ensamblan en homo o heterotrámeros entre miembros de una misma o una diferente subfamilia compartiendo mecanismos de regulación y tráfico, lo que resulta en una gran diversidad de conductancias cationicas, en términos de sus propiedades regulatorias y biofísicas, por lo que han sido descritos como sensores polimodales que responden a una gran variedad de estímulos intracelulares y externos, cumpliendo roles fisiológicos esenciales en las funciones sensoriales, homeostáticas e incluso diversas funciones motiles como la contracción muscular y la migración celular. TRPC3 es un miembro de la subfamilia TRPC que está involucrado en un amplio espectro de mecanismos de señalización de Ca2+, y presenta propiedades de activación y regulación únicas que le permiten el reconocimiento e integración de múltiples estímulos. Este canal se asocia con varias proteínas permitiendo la formación de canales catiónicos diferentes en diversos tipos de células, afectando su actividad y función, por lo que es considerado un sensor multifuncional y versátil de gran relevancia fisiológica y fisiopatológica. Se ha visto que TRPC3 interacciona físicamente con TRPM4 en sistemas de expresión heterólogos, un miembro de la subfamilia TRPM impermeable a calcio, pero activado por este catión, que está involucrado en diferentes procesos fisiológicos, y cuya ganancia de función está relacionada con gran variedad de eventos fisiopatológicos como cáncer, enfermedades cardiovasculares y neurodegenerativas. En nuestro laboratorio, se encontró que las proteínas End Binding (EBs) interaccionan con un motivo ‘SxIP’ ubicado en la región amino terminal de TRPM4, y que xiii esta interacción TRPM4-EB gobierna el tráfico anterógrado del canal y su actividad. De acuerdo a estos antecedentes, se propone validar la interacción física entre TRPC3 y TRPM4, y evaluar si es que comparten mecanismos de tráfico y exportación a la superficie celular. Para ello, se realizaron experimentos de inmunoprecipitación y se evaluó el co-trafico de estos canales utilizando diferentes aproximaciones en modelos de expresión heteróloga que coexpresaban ambos canales. Se encontró que efectivamente TRPM4 interacciona con TRPC3, y además que la deleción del ‘motivo SWIP’ de TRPM4 afecta la localización y exportación de TRPC3 a la superficie celular. Debido a la creciente relevancia fisiológica de los eventos de heteroasociación y co-trafico relacionados a los canales TRP, estos datos sugieren que la interacción física entre estos miembros de diferentes sufamilias TRP involucra mecanismos de tráfico asociado a la interacción TRPM4-EBs y podría tener relevancia en tejidos que coexpresen ambos canales, como en tejido cardiovascular o neuronal. / TRP proteins are non-selective cationic channels that permeate mainly Ca2+ and Na+, and share a general molecular architecture. TRP channels subunits assemble as homo or heterotetramers between members of the same or different subfamily, sharing regulation and trafficking mechanisms which results in a great diversity of cationic conductances, in terms of their regulatory and biophysical properties, so they have been described as polymodal sensors that respond to a wide variety of intracellular and external stimuli, fulfilling essential physiological roles in sensory, homeostatic and even diverse motile functions such as muscle contraction and cell migration. TRPC3 is a member of the TRPC subfamily involved in a broad spectrum of Ca2+ signaling mechanisms, and has unique activation and regulation properties that allow recognition and integration of multiple stimuli. These channels are associated with several proteins that allow it to form different cation channels in different types of cells, affecting the activity and function of the channel, so it is considered a multifunctional and versatile sensor of great physiological and physiopathological relevance. TRPC3 physically interacts with TRPM4, a member of the TRPM subfamily impermeable by calcium, but activated by this cation, which is involved in different physiological processes, and whose gain of function is related to a great variety of pathophysiological events, such as cancer, cardiovascular and neurodegenerative diseases. In our laboratory, we found that End Binding proteins (EBs) interact with a 'SxIP' motif located in the amino terminal region of TRPM4, and this TRPM4-EB interaction governs the anterograde trafficking of the channel and its activity. Accordingly, we achieved to validate the physical interaction between TRPC3 and TRPM4, and to evaluate whether these channels share trafficking and exporting mechanisms to the cell surface. To do that, we performed immunoprecipitation assays and the co-trafficking assays in heterologous systems that coexpressed both channels. We found that TRPM4 interacts with TRPC3. Moreover, we observed that the deletion of the 'SWIP motif' of TRPM4 affects the localization and exporting of TRPC3 to the cell surface. Due to the increasing physiological relevance of hetero-association and co-trafficking events related to TRP channels, these data suggest that the physical interaction between these members of different TRP subfamilies involve trafficking mechanisms associated with TRPM4-EBs interaction. These data could have relevance in tissues that coexpress both channels, as in cardiovascular or neuronal tissues. / Este Seminario de Título fue financiado por el proyecto FONDECYT 1160518 y Núcleo Milenio de Enfermedades Asociadas a Canales Iónicos (IR: Oscar Cerda A.),

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