Optogenetics has emerged as a branch of biotechnology that combines genetic engineering with optics to observe intracellular changes as well as control cellular function. Despite recent progress, there still remains the need for an optogenetic tool that can specifically control Ca2+. Such a tool would greatly facilitate the study of highly Ca2+-dependent cellular processes that are regulated both spatially and temporally. Ca2+ signaling regulates many cellular processes in both healthy and diseased cells. The ability to modulate the shape, duration, and amplitude of Ca2+ signaling is important for elucidating mechanisms by which endogenous Ca2+ concentrations are maintained. In this thesis, we used optogenetic approaches to explore a number of strategies to control Ca2+ influx through store-operated Ca2+ entry (SOCE) mediated by Stim1 and Orai1. To better study Ca2+ signaling in live cells, protein-based biosensors can be developed to monitor intracellular Ca2+ changes. To aid in this, we developed a computational modeling tool called FPMOD to improve both new and existing biosensor designs. Although FPMOD was initially intended for evaluating biosensor designs, other research groups have since used it to construct models of other proteins to answer questions related to protein conformation. We next studied the modulation of SOCE by using drug-inducible fusion proteins to study the regulation of Stim1 puncta formation. Interestingly, recruiting a Ca2+-buffering protein to Stim1 led to puncta formation, a previously unknown means of inducing puncta. These results suggest Stim1 may additionally be regulated by cytoplasmic Ca2+ levels. Finally, we developed LOVS1K, an optogenetic tool to directly activate Orai1 channels and specifically control Ca2+ influx. Photo-sensitive LOVS1K was used to generate both local Ca2+ influx at the membrane and global cytoplasmic Ca2+ signals. As proof of concept, LOVS1K was further used to modulate engineered Ca2+-dependent proteins. Ca2+ is a remarkably versatile intracellular messenger. The combination of high spatiotemporal control of irradiation and the ability of LOVS1K to generate both local and global Ca2+ changes provides a promising platform to study cellular processes that are highly dependent on different Ca2+ signals. Together, biosensors and engineered Ca2+-modulating tools can be used to study the many different aspects of Ca2+ signaling and controllably manipulate endogenous Ca2+ signaling pathways.

Optogenetics has emerged as a branch of biotechnology that combines genetic engineering with optics to observe intracellular changes as well as control cellular function. Despite recent progress, there still remains the need for an optogenetic tool that can specifically control Ca2+. Such a tool would greatly facilitate the study of highly Ca2+-dependent cellular processes that are regulated both spatially and temporally. Ca2+ signaling regulates many cellular processes in both healthy and diseased cells. The ability to modulate the shape, duration, and amplitude of Ca2+ signaling is important for elucidating mechanisms by which endogenous Ca2+ concentrations are maintained. In this thesis, we used optogenetic approaches to explore a number of strategies to control Ca2+ influx through store-operated Ca2+ entry (SOCE) mediated by Stim1 and Orai1. To better study Ca2+ signaling in live cells, protein-based biosensors can be developed to monitor intracellular Ca2+ changes. To aid in this, we developed a computational modeling tool called FPMOD to improve both new and existing biosensor designs. Although FPMOD was initially intended for evaluating biosensor designs, other research groups have since used it to construct models of other proteins to answer questions related to protein conformation. We next studied the modulation of SOCE by using drug-inducible fusion proteins to study the regulation of Stim1 puncta formation. Interestingly, recruiting a Ca2+-buffering protein to Stim1 led to puncta formation, a previously unknown means of inducing puncta. These results suggest Stim1 may additionally be regulated by cytoplasmic Ca2+ levels. Finally, we developed LOVS1K, an optogenetic tool to directly activate Orai1 channels and specifically control Ca2+ influx. Photo-sensitive LOVS1K was used to generate both local Ca2+ influx at the membrane and global cytoplasmic Ca2+ signals. As proof of concept, LOVS1K was further used to modulate engineered Ca2+-dependent proteins. Ca2+ is a remarkably versatile intracellular messenger. The combination of high spatiotemporal control of irradiation and the ability of LOVS1K to generate both local and global Ca2+ changes provides a promising platform to study cellular processes that are highly dependent on different Ca2+ signals. Together, biosensors and engineered Ca2+-modulating tools can be used to study the many different aspects of Ca2+ signaling and controllably manipulate endogenous Ca2+ signaling pathways.

No description available.

Ca2+ is an important mediator of stimulus-secretion coupling in beta cells of the pancreatic islets, which secrete insulin in response to elevation in plasma glucose concentration. I studied the actions of two lipids, arachidonic acid (AA) and cholesterol, on enzymatically-dissociated single beta cells of rat and mouse, using cytosolic Ca2+ ([Ca2+]i) measurement in conjunction with whole-cell patch-clamp techniques. AA, which is produced in the beta cell upon stimulation with either glucose or acetylcholine, was found to induce a large increase in [Ca2+]i that was dependent on both extracellular Ca2+ entry and intracellular Ca2+ release. Part of the AA-mediated extracellular Ca2+ entry was due to Ca2+ influx through the arachidonate-regulated Ca2+ (ARC) channels, which have not previously been reported in beta cells. The AA-mediated intracellular Ca2+ release was a result of Ca2+ mobilization from multiple inositol trisphosphate (IP3)-sensitive intracellular stores, including the endoplasmic reticulum (ER) and an acidic Ca2+ store that is probably the secretory granules. Therefore, in beta cells, the AA-mediated Ca2+ signal may amplify the [Ca2+]i rise induced by insulin secretagogues. Cholesterol is an integral component of cellular membranes and an important regulator of cellular functions. However, elevation of cholesterol level in the pancreatic islets reduces glucose-stimulated insulin secretion. I found that cholesterol overload impairs the glucose-stimulated [Ca2+]i increase in beta cells by two major mechanisms: the first is a decrease in glucose-stimulated ATP production, which is partly mediated by a decrease in glucose uptake, and the second is the reduction of voltage-gated Ca2+ current density. These effects of cholesterol may partly account for the decreased insulin secretion that develops in patients with type II diabetes, who typically exhibit hypercholesterolemia. In summary, different lipids may mediate beneficial or detrimental effects on Ca2+ regulation in rodent pancreatic beta cells.

Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.

No description available.

Store-operated calcium entry (SOCE) is a principal mechanism for extracellular calcium entry in non-excitable cell types, and is primarily facilitated by the calcium- release activated calcium (CRAC) channel; itself comprised of the pore-forming Orai-1 and calcium-sensing Stromal interaction molecule (STIM)-1 proteins. Depletion of endoplasmic reticulum (ER) calcium stores initiates STIM-1 translocation to defined ER-plasma membrane puncta, and subsequent Orai-STIM interaction and opening of Orai. The importance of this mechanism in calcium signalling in diverse tissue types is becoming increasingly clear. The vascular endothelium is a dynamic tissue, involved in the maintenance of vascular homeostasis and haemostasis. Many endothelium-derived bioactive agents, such as endothelin-1, prostaglandins, and the potent vasodilator nitric oxide (NO), are known to be produced via calcium- dependent mechanisms. However, the role of the CRAC channel in the vascular endothelium is poorly defined with little known about downstream targets of calcium influx through CRAC channels. The dysregulation of NO production by endothelial nitric oxide synthase (eNOS) is a major contributory factor in many vascular disease states, yet the calcium channel responsible for eNOS activation has yet to be identified. Within this thesis, I establish the endothelial cell line sEnd.1 as a new model system for studying CRAC channel signalling in the vascular endothelium, defining sEnd.1 SOCE as being CRAC channel-dependent. Inhibition of CRAC channels with an array of inhibitors, and knock-down of STIM-1, both reduced ATP- and TG-induced SOCE. The sEnd.1 model system was subsequently used to identify calcium entry through the CRAC channel as the elusive activation mechanism for eNOS. Through real-time imaging with the fluorescent NO dye DAF-2-DA, we established that NO production is non-linear, with a slow initial increase preceding a faster NO production phase. These kinetics, with a characteristic delay before fast production have, to our knowledge, not previously been reported. The time taken to reach the fast phase of NO production could be manipulated through changes in both local and bulk calcium rises, which indicated roles for both elements of calcium signalling in eNOS activation. eNOS regulation by calcium is complex, occurring not only through direct binding of calcium-calmodulin, but additionally through changing post-translational modifications, which in turn regulate the calcium-dependency of eNOS, such as phosphorylation of Ser1177. We propose that the delay in fast production of NO is due to the time taken to alter eNOS post-translational modifications, which thus remove inhibition on eNOS. Activation of CRAC channels increased phosphorylation of residue Ser1177 via calcium-calmodulin kinase II (CaMKII), with a similar time course to that required to reach the fast phase of NO production. Inhibition of CaMKII increased the time taken to reach fast activation. In conclusion this thesis presents a new model system for investigation of CRAC channel signalling in the endothelium. Furthermore, we clearly identify a critical endothelial pathway as being regulated by CRAC channels, by demonstrating the production of NO in response to both ATP and TG, which stimulate calcium entry through CRAC channels.

<p><strong>Introduction</strong></p> <p>Asthma is a chronic respiratory disease that is becoming more prevalent. Airway hyperresponsivness, a key feature of asthma, involves increased narrowing of the airways in response to bronchoconstricting agents. Airway smooth muscle (ASM) functioning is largely responsible for hyperresponsiveness yet the mechanisms behind excitation-contraction coupling are not fully understood. Src tyrosine kinase contributes to contraction in other smooth muscle types. Furthermore, STIM1, Orai1, IPLA₂b and RyRs play a role in ASM excitation-contraction coupling.</p> <p><strong>Aim</strong></p> <p>We sought to determine whether Src activity is involved in serotonin (5-HT)- and acetylcholine (ACh)-induced ASM contraction. We also examined whether the gene expression of molecules involved in sarcoplasmic reticulum emptying and refilling is altered during airway hyperresponsiveness.</p> <p><strong>Methods</strong></p> <p>Bovine tracheal ASM strips were pre-treated with the non-specific tyrosine kinase inhibitor genistein (10^-4M), src kinase family inhibitors PP1 (10^-5M) and PP2 (10^-5M) or vehicle and challenged with either 5-HT or ACh to determine the involvment of Src in contraction. Western blotting was used to examine Src activity following 5-HT or ACh treatment. Female BALB/c mice were exposed to an intranasal injection of [1.7mg/ml] HDM extract or saline. Real time, reverse-transcriptase polymerase chain reaction was used to examine gene expression.</p> <p><strong> </strong></p> <p><strong>Results</strong></p> <p>Genistein, PP1 and PP2 significantly reduced 5-HT-induced ASM contractions and Src activity was significantly increased in response to 5-HT. ACh-induced contractions were significantly reduced by genistein, but not PP1 and PP2. However, Src activity was significantly increased by ACh. RyR3 mRNA expression was significantly increased, Orai1 was significantly decreased, and STIM1, IPLA₂b, RyR1 and RyR2 were unchanged by the house dust mite treatment.</p> <p><strong>Conclusion</strong></p> <p>These data suggets 5-HT-induced ASM contraction involves Src activity. However, ACh-induced ASM contractions might not require Src. The changes in RyR3 and Orai1 expression might alter Ca²⁺-handling in such a way as to potentiate airway hyperresponsiveness but further investigation is required.</p> / Master of Science (MSc)

Les polynucléaires neutrophiles représentent 50-70% des leucocytes sanguins et possèdent un rôle majeur dans la défense de l’organisme contre les pathogènes. Le Ca2+ est un second messager qui joue un rôle primordial dans le chimiotactisme, la phagocytose, la dégranulation et la production de formes réactives de l’oxygène (FRO) afin de neutraliser l’agent pathogène. Dans ces cellules, l’influx calcique de type SOCE est essentiel pour l'homéostasie calcique. Il est peu étudié en raison du manque d’outils pharmacologiques spécifiques d’où l’importance dans un premier temps de chercher de nouvelles molécules. Les cellules T Jurkat dont le SOCE est largement caractérisé servent de modèle pour la caractérisation initiale de ces molécules. Le 2-APB est parmi les molécules les plus largement utilisées dans la caractérisation du SOCE en raison de sa double activité sur le SOCE avec une potentialisation à [1-10 μM] et une inhibition à [> 20 μM]. En revanche, ce produit manque de spécificité et agit sur d’autres cibles cellulaires comme les récepteurs à l’inositol (1,4,5)-trisphosphate (InsP3Rs). La 1ère étape est de sélectionner à partir d’analogues commerciaux du 2-APB (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB et MDEB), des composés plus spécifiques et également plus efficaces que la molécule mère. Deux molécules se sont distinguées : le MDEB comme uniquement potentialisant du SOCE et le Benzothienyl-APB comme un puissant inhibiteur. En revanche, tous les analogues du 2-APB inhibent les InsP3Rs à l’exception du MDEB qui semble plus spécifique du SOCE. L’effet du MDEB sur le courant calcique, ICRAC, a été étudié grâce à la technique du patch-clamp. Il augmente d’environ 4 fois l’amplitude de ICRAC par rapport à celle enregistrée dans les cellules contrôle. Par ailleurs, le MDEB ralentie l’inactivation rapide de ICRAC due au Ca2+. Sur le plan physiologique, le MDEB à des concentrations croissantes inhibe la synthèse de l’IL-2 dans les cellules Jurkat stimulées et ceci malgré son effet potentialisant du SOCE. Cette activité est liée à son effet pro-apoptotique dans les cellules Jurkat stimulées. Le MDEB et le Benzothienyl-APB caractérisés dans la 1ère partie nous ont servi d’outils potentiels afin d’étudier le SOCE des cellules PLB-985 différenciées en cellules proches de neutrophiles. Le SOCE a été induit soit par un traitement des cellules avec la thapsigargine (Tg) soit de manière physiologique avec les peptides fMLF et le WKYMVm deux chimioattractants, ligands des récepteurs aux peptides formylés FPR et FPRL1 respectivement. En plus, le SOCE induit par la Tg est modulable par le 2-APB, potentialisé par le MDEB et inhibé par le Benzothienyl-APB. La phagocytose des levures par les cellules PLB-985 différenciées ainsi que la production de FRO intraphagosomales ont été inhibées par le MDEB et le Benzothienyl-APB. Les FRO extracellulaires ont été également inhibées par Benzothienyl-APB en revanche à cause de la forte interférence du MDEB avec la technique de mesure nous n’avons pas pu étudier ses activités. En conclusion, le MDEB et le Benzothienyl-APB sont de nouveaux outils pharmacologiques potentialisant ou inhibant le SOCE des leucocytes, qui nous permettront dans l’avenir une meilleure compréhension de l'entrée calcique et ses rôles dans ces cellules. / Neutrophils represent 50-70% of human blood leukocytes; their role is to protect the body against pathogens. Calcium is a second messenger which plays an important role in chemotaxis, phagocytosis, degranulation and the production of reactive oxygen species (ROS) in order to eliminate microbes. In neutrophils, the mechanism of store-operated calcium entry (SOCE) is essential for calcium homeostasis. However, neutrophil SOCE is not well understood because of the lack of specific pharmacological tools. It is necessary to first identify and characterize new molecules using a model of Jurkat T cells in which SOCE was the best characterized. 2-APB is the most widely used molecule in SOCE characterization due to its dual activity with a potentiation at lower concentrations [1-10 μM] and an inhibition at higher concentrations [> 20 μM]. However, this molecule lacks specificity because it acts on other cellular targets such as inositol (1,4,5)-trisphosphate receptors (InsP3Rs). The first step is to select from a library of 8 commercial 2-APB analogues (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB and MDEB) those that are more specific and also more efficient molecules than 2-APB. Two interesting molecules were identified, MDEB as the only SOCE potentiating product currently known and the Benzothienyl-APB, which is a strong inhibitor. Like 2-APB, all these analogues inhibit InsP3Rs except MDEB, which seems to be more specific. The effect of MDEB on the calcium current, ICRAC, was also studied using the patch-clamp technique. MDEB increases ~4 times the ICRAC amplitude in comparison with control. Otherwise, MDEB slows down the fast Ca2 +-dependent inactivation of ICRAC. Functionally, MDEB at increasing concentrations inhibits IL-2 synthesis in stimulated Jurkat T cells despite its potentiating activity on SOCE. The inhibition is due to MDEB induced apoptosis in stimulated Jurkat T cells. MDEB and Benzothienyl-APB were then used as tools to study SOCE in a neutrophil-like cell model, the differentiated PLB-985 cells. SOCE was induced either by treatment of cells with thapsigargin (Tg) or physiologically with the chemotactic peptides fMLF and WKYMVm, ligands of formyl peptide receptors FPR and FPRL1 respectively. In addition, Tg-induced SOCE was modulated by 2-APB, potentiated by MDEB and inhibited by Benzothienyl-APB. The consequences of these analogues on neutrophil functions were also studied. Phagocytosis of yeasts by PLB-985 cells and intraphagosomal ROS production were inhibited by MDEB and Benzothienyl-APB. Furthermore, extracellular ROS were also inhibited by Benzothienyl-APB. However, because of the high interference of MDEB with our techniques, its activities could not be studied. In conclusion, MDEB and Benzothienyl-APB are new analogues of 2-APB potentiating and inhibiting SOCE, which allow us in the future a better understanding of leukocyte SOCE and its cellular roles.

La neteja del moc i dels patògens dels pulmons, i el transport de gàmets i embrions en els òrgans reproductius de les femelles són funcions clau en els epitelis ciliats, tals com aquells que es troben presents en les vies respiratòries i l'oviducte. La taxa de transport mucociliar és funció de la freqüència de batut ciliar (CBF) i aquesta freqüència és augmentada per increments en la concentració de Ca2+ intracelul·lar. El canal catiònic "transient potential vanilloid 4" (TRPV4) intervé en l'entrada de Ca2+ en resposta a estímuls mecànics i osmòtics. L'expressió del TRPV4 en l'epiteli ciliat de les vies respiratòries i de l'oviducte és confirmada mitjançant la localització per immunofluorescència del canal iònic a la membrana apical de l'epiteli ciliat i polaritzat, allà on la senyalització de Ca2+ és requerida per la regulació de la CBF. Cèl·lules ciliades de la tràquea de ratolins TRPV4-/- no expressen el canal TRPV4, no responen a l'activador específic del TRPV4, el 4&#945;-phorbol 12,13-didecanoate (4&#945;-PDD) i presenten respostes de Ca2+ reduïdes a temperatures mitjanes (~25ºC- 8ºC), un altre estímul dels canals TRPV4. L'activació dels canals TRPV4 per solucions altament viscoses i per hypotonicitat depèn de l'activació de la via de la fosfolipasa A2(PLA2)i la subseqüent producció de àcid epoxieicosatrienoic (EET). En condicions de baixa activació de la PLA2, estímuls mecànics i hipotònics alliberen ATP per a l'activació de la via de la fosfolipasa C (PLC)-inositol trifosfat (IP3) per contribuir a l'activació dels canals TRPV4. Descrivim que el metabòlit IP3 sense ser un agonista per ell mateix, sensibilitza el TRPV4 per a l'activació de EET, essent aquest un mecanisme general. L'acoblament funcional entre els canals TRPV4 de la membrana plasmàtica i els receptors de IP3 (IP3R) és necessari tant per iniciar com mantenir la senyalització oscil·latòria del Ca2+ desencadenada per estímuls viscosos i hipotònics. Un dels principals activadors de la CBF, la adenosina-5'-trifosfat (ATP), desencadena una resposta cel·lular mediada per Ca2+ en la que es desencadena tant l'alliberament de Ca2+ des dels dipòsits intracel·lulars com l'entrada de Ca2+. És destacable la contribució de el TRPV4 en l'augment de la CBF mediada per ATP. És més, el nostre treball implica als canals TRPV4 exclusivament en l'entrada de Ca2+ activada per receptor (ROCE). Tot plegat, aquesta tesi doctoral mostra el paper dels canals TRPV4 en l'acoblament d'estímuls fisiològics tipus mecànic, osmòtic i químic a la regulació de la CBF en l'epiteli ciliat destinat al transport mucociliar. / Clearance of mucus and pathogenic agents from lungs and the transport of gametes and embryos in the female reproductive organs are key functions of ciliated epithelia such as those present in the airways and the oviduct. The rate of mucociliary transport is a function of ciliary beat frequency (CBF) and this, in turn, is increased by increases in intracellular calcium. Transient potential vanilloid 4 (TRPV4)cation channel mediates Ca2+ influx in response to mechanical and osmotic stimuli. TRPV4 expression in ciliated epithelia from airways and oviduct is confirmed by immunofluorescence localization of the channel at the apical membrane of the polarized ciliated epithelia, where the Ca2+ signalling is required for CBF regulation. Ciliated tracheal cells from TRPV4-/-mice show no TRPV4 expression, neither increases in intracellular Ca2+ and CBF in response to the TRPV4-specific activator 4&#945;- phorbol 12,13- idecanoate (4&#945;-PDD), and reduced responses to mild temperatures (~25ºC - 38ºC), another TRPV4-activating stimulus. TRPV4 gating by high viscous loads and hypotonicity depends on phospholipase A2 (PLA2) pathway activation and subsequent production of epoxyeicosatrienoic acid (EET). Under conditions of low PLA2 activation, mechanical and hypotonic stimuli use extracellular ATP release-mediated activation of phospholipase C (PLC)-inositol triphosphate(IP3)signalling to support TRPV4 gating. We describe that IP3, without being an agonist itself, sensitizes TRPV4 to EET activation. Besides, the functional coupling between plasma membrane TRPV4 channels and IP3 receptors (IP3R) is required to initiate and maintain the cellular oscillatory Ca2+ signal triggered by high viscous loads and hypotonic stimuli. One of the main CBF activators, adenosine-5'-triphosphate (ATP), triggers both Ca2+ release from intracellular Ca2+ stores and Ca2+ entry. Interestingly, TRPV4 contributes to ATP-induced increase in CBF. Furthermore, our work implicates TRPV4 channel exclusively in receptor-operated Ca2+ entry. Collectively, this PhD thesis shows the role of TRPV4 channels coupling physiologically relevant mechanical, hypotonic and chemical stimuli to CBF regulation in motile ciliary epithelia.