{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/374317"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/374317","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Dual regulation of inositol 1,4,5-trisphosphate receptors by inositol 1,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate","abstract":"Ca2+ is a universal and effective intracellular messenger, which holds a central role in the regulation of a vast array of cellular processes. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are key signal integrators, which transform extracellular stimuli into intracellular Ca2+ signals. Only immobilised IP3Rs, licensed by association with KRas-induced actin-interacting protein (KRAP), can respond through IP3-mediated Ca2+ release. These licensed IP3Rs are tethered on actin near membrane contact sites (MCS) between the endoplasmic reticulum (ER) and plasma membrane (PM), where store-operated Ca2+ entry (SOCE) takes place. Uncovering the mechanisms that govern IP3R regulation is an essential element in understanding the spatial and temporal patterns of Ca2+ signalling. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor, but functionally diverse lipid at the PM. In the canonical Ca2+ signalling cascade, activation of PM-resident receptors such as G-protein coupled receptors (GPCRs) causes phospholipase C (PLC) to hydrolyse PI(4,5)P2, producing inositol 1,4,5-trisphosphate (IP3) which diffuses through the cytoplasm to IP3Rs, enabling Ca2+ release from the ER. The stimulus intensity governs the amount of IP3 produced and in consequence, the extent of IP3R activation, starting from brief, localised Ca2+ puffs, and progressing to cell-wide global Ca2+ waves. Tight control of the transition from local to global Ca2+ signals is central to the downstream consequences of receptor activation. PI(4,5)P2, with its essential roles in F-actin nucleation and formation of the SOCE complex at ER-PM MCS, is thus a potential regulator of IP3R activity in addition to its role in providing IP3. Towards exploring possible additional roles of PI(4,5)P2 in regulating Ca2+ signalling via IP3Rs, I have assessed three strategies for selective depletion of PI(4,5)P2 at the PM: pharmacological inhibition of the synthesis of phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2, gene silencing of the 5-kinases that convert PI(4)P to PI(4,5)P2, and expression of a rapamycin-inducible heterodimerization system that allows translocation of a PI(4,5)P2-specific 5-phosphatase (herein referred to as the ‘’5-PTASE system’’) to the PM. Pharmacological or siRNA-mediated inhibition of the relevant kinases in the PI(4,5)P2 metabolic cycle did not successfully attenuate Ca2+ signals in response to histamine in HeLa cells, suggesting that PLC-sensitive PI(4,5)P2 pools remain available after treatment. On the contrary, I have shown that the 5-PTASE system caused global depletion of PI(4,5)P2 at the PM using a genetically encoded, PI(4,5)P2-selective fluorescent sensor, and showed that Ca2+ signals in response to histamine in HeLa cells were attenuated. The method thereby allows acute and near-complete depletion of PM-associated PI(4,5)P2. Using the validated 5-PTASE system for PM PI(4,5)P2 depletion, and uniform delivery of i-IP3 to the cytosol via uncaging of the exogenously supplied photolabile caged ci-IP3, revealed that PI(4,5)P2 depletion significantly reduced the frequency of Ca2+ puffs in HeLa and HEK293 cells without affecting puff amplitude or kinetics. PI(4,5)P2 regulation was confirmed to extend to all three IP3R subtypes. As PI(4,5)P2 depletion may lead to reduction of basal IP3 levels, I employed two complementary approaches to assess whether a loss of basal IP3 is responsible for the reduced Ca2+ puff frequency. Reducing basal IP3 levels by inhibiting PLC activity with U73122 or by overexpressing cytosolic IP3 kinase C (IP3KC) did not reduce the frequency of Ca2+ puffs evoked by photolysis of ci-IP3. I conclude that PI(4,5)P2 regulates IP3R activity in parallel to providing IP3. As PI(4,5)P2 levels at the PM are dynamically controlled during signalling when PI(4,5)P2 is consumed to produce IP3, I developed methods to uncouple stimulation of GPCRs that evoke IP3 formation from delivery of IP3 to IP3Rs while retaining opportunities to stimulate IP3Rs directly. I expressed IP3KC to intercept endogenous IP3 and photolyzed ci-IP3 to enable independent activation of GPCRs and delivery of i-IP3 to IP3Rs. Activation of H1 receptors in HeLa cells or M3 muscarinic receptors in HEK293 cells in the presence of IP3KC reduced the frequency of Ca2+ puffs evoked exogenously by photolysis of ci-IP3 without affecting puff amplitude or kinetics. This inhibition was entirely mediated by PI(4,5)P2 depletion. PI(4,5)P2 depletion significantly reduced the likelihood of Ca2+ puffs progressing to a global Ca2+ signal, but once the transition threshold was reached, the amplitude of the global signal was indistinguishable in the presence and absence of PI(4,5)P2. PI(4,5)P2 depletion did not affect the subcellular distribution of IP3Rs. I suggest that PI(4,5)P2 primes IP3Rs to respond to IP3 by partially occupying the receptor’s IP3-binding site. Increasing PI(4,5)P2 levels in the PM did not further activate IP3Rs, suggesting that basal PI(4,5)P2 achieves the maximal effect. It is unclear whether this occurs with all IP3-binding sites occupied by PI(4,5)P2 or whether physical barriers constrain the number of sites that can be occupied. My results establish that PI(4,5)P2 primes IP3Rs to respond, and that as GPCRs stimulate IP3 formation they also deplete PI(4,5)P2, relieving this priming stimulus and resetting IP3R sensitivity. Dual regulation of IP3Rs by PI(4,5)P2 and IP3 through GPCRs thus controls the transition from local to global Ca2+ signals.","abstract_html":"Ca<sup>2+</sup> is a universal and effective intracellular messenger, which holds a central role in the regulation of a vast array of cellular processes. Inositol 1,4,5-trisphosphate receptors (IP<sub>3</sub>Rs) are key signal integrators, which transform extracellular stimuli into intracellular Ca<sup>2+</sup> signals. Only immobilised IP<sub>3</sub>Rs, licensed by association with KRas-induced actin-interacting protein (KRAP), can respond through IP<sub>3</sub>-mediated Ca<sup>2+</sup> release. These licensed IP<sub>3</sub>Rs are tethered on actin near membrane contact sites (MCS) between the endoplasmic reticulum (ER) and plasma membrane (PM), where store-operated Ca<sup>2+</sup> entry (SOCE) takes place. Uncovering the mechanisms that govern IP<sub>3</sub>R regulation is an essential element in understanding the spatial and temporal patterns of Ca<sup>2+</sup> signalling. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P<sub>2</sub>) is a minor, but functionally diverse lipid at the PM. In the canonical Ca<sup>2+</sup> signalling cascade, activation of PM-resident receptors such as G-protein coupled receptors (GPCRs) causes phospholipase C (PLC) to hydrolyse PI(4,5)P<sub>2</sub>, producing inositol 1,4,5-trisphosphate (IP<sub>3</sub>) which diffuses through the cytoplasm to IP<sub>3</sub>Rs, enabling Ca<sup>2+</sup> release from the ER. The stimulus intensity governs the amount of IP<sub>3</sub> produced and in consequence, the extent of IP<sub>3</sub>R activation, starting from brief, localised Ca<sup>2+</sup> puffs, and progressing to cell-wide global Ca<sup>2+</sup> waves. Tight control of the transition from local to global Ca<sup>2+</sup> signals is central to the downstream consequences of receptor activation. PI(4,5)P<sub>2</sub>, with its essential roles in F-actin nucleation and formation of the SOCE complex at ER-PM MCS, is thus a potential regulator of IP<sub>3</sub>R activity in addition to its role in providing IP<sub>3</sub>. Towards exploring possible additional roles of PI(4,5)P<sub>2</sub> in regulating Ca<sup>2+</sup> signalling via IP<sub>3</sub>Rs, I have assessed three strategies for selective depletion of PI(4,5)P<sub>2</sub> at the PM: pharmacological inhibition of the synthesis of phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P<sub>2</sub>, gene silencing of the 5-kinases that convert PI(4)P to PI(4,5)P<sub>2</sub>, and expression of a rapamycin-inducible heterodimerization system that allows translocation of a PI(4,5)P<sub>2</sub>-specific 5-phosphatase (herein referred to as the ‘’5-PTASE system’’) to the PM. Pharmacological or siRNA-mediated inhibition of the relevant kinases in the PI(4,5)P<sub>2</sub> metabolic cycle did not successfully attenuate Ca<sup>2+</sup> signals in response to histamine in HeLa cells, suggesting that PLC-sensitive PI(4,5)P<sub>2</sub> pools remain available after treatment. On the contrary, I have shown that the 5-PTASE system caused global depletion of PI(4,5)P<sub>2</sub> at the PM using a genetically encoded, PI(4,5)P<sub>2</sub>-selective fluorescent sensor, and showed that Ca<sup>2+</sup> signals in response to histamine in HeLa cells were attenuated. The method thereby allows acute and near-complete depletion of PM-associated PI(4,5)P<sub>2</sub>. Using the validated 5-PTASE system for PM PI(4,5)P<sub>2</sub> depletion, and uniform delivery of i-IP<sub>3</sub> to the cytosol via uncaging of the exogenously supplied photolabile caged ci-IP<sub>3</sub>, revealed that PI(4,5)P<sub>2</sub> depletion significantly reduced the frequency of Ca<sup>2+</sup> puffs in HeLa and HEK293 cells without affecting puff amplitude or kinetics. PI(4,5)P<sub>2</sub> regulation was confirmed to extend to all three IP<sub>3</sub>R subtypes. As PI(4,5)P<sub>2</sub> depletion may lead to reduction of basal IP<sub>3</sub> levels, I employed two complementary approaches to assess whether a loss of basal IP<sub>3</sub> is responsible for the reduced Ca<sup>2+</sup> puff frequency. Reducing basal IP<sub>3</sub> levels by inhibiting PLC activity with U73122 or by overexpressing cytosolic IP<sub>3</sub> kinase C (IP<sub>3</sub>KC) did not reduce the frequency of Ca<sup>2+</sup> puffs evoked by photolysis of ci-IP<sub>3</sub>. I conclude that PI(4,5)P<sub>2</sub> regulates IP<sub>3</sub>R activity in parallel to providing IP<sub>3</sub>. As PI(4,5)P<sub>2</sub> levels at the PM are dynamically controlled during signalling when PI(4,5)P<sub>2</sub> is consumed to produce IP<sub>3</sub>, I developed methods to uncouple stimulation of GPCRs that evoke IP<sub>3</sub> formation from delivery of IP<sub>3</sub> to IP<sub>3</sub>Rs while retaining opportunities to stimulate IP<sub>3</sub>Rs directly. I expressed IP<sub>3</sub>KC to intercept endogenous IP<sub>3</sub> and photolyzed ci-IP<sub>3</sub> to enable independent activation of GPCRs and delivery of i-IP<sub>3</sub> to IP<sub>3</sub>Rs. Activation of H1 receptors in HeLa cells or M3 muscarinic receptors in HEK293 cells in the presence of IP<sub>3</sub>KC reduced the frequency of Ca<sup>2+</sup> puffs evoked exogenously by photolysis of ci-IP<sub>3</sub> without affecting puff amplitude or kinetics. This inhibition was entirely mediated by PI(4,5)P<sub>2</sub> depletion. PI(4,5)P<sub>2</sub> depletion significantly reduced the likelihood of Ca<sup>2+</sup> puffs progressing to a global Ca<sup>2+</sup> signal, but once the transition threshold was reached, the amplitude of the global signal was indistinguishable in the presence and absence of PI(4,5)P<sub>2</sub>. PI(4,5)P<sub>2</sub> depletion did not affect the subcellular distribution of IP<sub>3</sub>Rs. I suggest that PI(4,5)P<sub>2</sub> primes IP<sub>3</sub>Rs to respond to IP<sub>3</sub> by partially occupying the receptor’s IP<sub>3</sub>-binding site. Increasing PI(4,5)P<sub>2</sub> levels in the PM did not further activate IP<sub>3</sub>Rs, suggesting that basal PI(4,5)P<sub>2</sub> achieves the maximal effect. It is unclear whether this occurs with all IP<sub>3</sub>-binding sites occupied by PI(4,5)P<sub>2</sub> or whether physical barriers constrain the number of sites that can be occupied. My results establish that PI(4,5)P<sub>2</sub> primes IP<sub>3</sub>Rs to respond, and that as GPCRs stimulate IP<sub>3</sub> formation they also deplete PI(4,5)P<sub>2</sub>, relieving this priming stimulus and resetting IP<sub>3</sub>R sensitivity. Dual regulation of IP<sub>3</sub>Rs by PI(4,5)P<sub>2</sub> and IP<sub>3</sub> through GPCRs thus controls the transition from local to global Ca<sup>2+</sup> signals.","abstract_has_math":false,"creators":["Ivanova, Adelina Adelin"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Ladds, Graham"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-06-08","date_published":"2024-06-08","updated_at":"2026-07-22T22:24:24Z","subjects":["Ca2+","endoplasmic reticulum","IP3R","PI(4,5)P2","rapamycin","signalling"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/85d0f03d-ef0e-4e8e-b460-802ab1fc510c/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.112415","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Ladds, Graham"]},{"key":"dc:creator","label":"Author","values":["Ivanova, Adelina Adelin"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-06-08"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/374317"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Ca2+","endoplasmic reticulum","IP3R","PI(4,5)P2","rapamycin","signalling"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/85d0f03d-ef0e-4e8e-b460-802ab1fc510c/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.112415"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/37edb92f-4a98-4b67-8e41-fb1da0cb17e5/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Ca2+ is a universal and effective intracellular messenger, which holds a central role in the regulation of a vast array of cellular processes. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are key signal integrators, which transform extracellular stimuli into intracellular Ca2+ signals. Only immobilised IP3Rs, licensed by association with KRas-induced actin-interacting protein (KRAP), can respond through IP3-mediated Ca2+ release. These licensed IP3Rs are tethered on actin near membrane contact sites (MCS) between the endoplasmic reticulum (ER) and plasma membrane (PM), where store-operated Ca2+ entry (SOCE) takes place. Uncovering the mechanisms that govern IP3R regulation is an essential element in understanding the spatial and temporal patterns of Ca2+ signalling. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor, but functionally diverse lipid at the PM. In the canonical Ca2+ signalling cascade, activation of PM-resident receptors such as G-protein coupled receptors (GPCRs) causes phospholipase C (PLC) to hydrolyse PI(4,5)P2, producing inositol 1,4,5-trisphosphate (IP3) which diffuses through the cytoplasm to IP3Rs, enabling Ca2+ release from the ER. The stimulus intensity governs the amount of IP3 produced and in consequence, the extent of IP3R activation, starting from brief, localised Ca2+ puffs, and progressing to cell-wide global Ca2+ waves. Tight control of the transition from local to global Ca2+ signals is central to the downstream consequences of receptor activation. PI(4,5)P2, with its essential roles in F-actin nucleation and formation of the SOCE complex at ER-PM MCS, is thus a potential regulator of IP3R activity in addition to its role in providing IP3. Towards exploring possible additional roles of PI(4,5)P2 in regulating Ca2+ signalling via IP3Rs, I have assessed three strategies for selective depletion of PI(4,5)P2 at the PM: pharmacological inhibition of the synthesis of phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2, gene silencing of the 5-kinases that convert PI(4)P to PI(4,5)P2, and expression of a rapamycin-inducible heterodimerization system that allows translocation of a PI(4,5)P2-specific 5-phosphatase (herein referred to as the ‘’5-PTASE system’’) to the PM. Pharmacological or siRNA-mediated inhibition of the relevant kinases in the PI(4,5)P2 metabolic cycle did not successfully attenuate Ca2+ signals in response to histamine in HeLa cells, suggesting that PLC-sensitive PI(4,5)P2 pools remain available after treatment. On the contrary, I have shown that the 5-PTASE system caused global depletion of PI(4,5)P2 at the PM using a genetically encoded, PI(4,5)P2-selective fluorescent sensor, and showed that Ca2+ signals in response to histamine in HeLa cells were attenuated. The method thereby allows acute and near-complete depletion of PM-associated PI(4,5)P2. Using the validated 5-PTASE system for PM PI(4,5)P2 depletion, and uniform delivery of i-IP3 to the cytosol via uncaging of the exogenously supplied photolabile caged ci-IP3, revealed that PI(4,5)P2 depletion significantly reduced the frequency of Ca2+ puffs in HeLa and HEK293 cells without affecting puff amplitude or kinetics. PI(4,5)P2 regulation was confirmed to extend to all three IP3R subtypes. As PI(4,5)P2 depletion may lead to reduction of basal IP3 levels, I employed two complementary approaches to assess whether a loss of basal IP3 is responsible for the reduced Ca2+ puff frequency. Reducing basal IP3 levels by inhibiting PLC activity with U73122 or by overexpressing cytosolic IP3 kinase C (IP3KC) did not reduce the frequency of Ca2+ puffs evoked by photolysis of ci-IP3. I conclude that PI(4,5)P2 regulates IP3R activity in parallel to providing IP3. As PI(4,5)P2 levels at the PM are dynamically controlled during signalling when PI(4,5)P2 is consumed to produce IP3, I developed methods to uncouple stimulation of GPCRs that evoke IP3 formation from delivery of IP3 to IP3Rs while retaining opportunities to stimulate IP3Rs directly. I expressed IP3KC to intercept endogenous IP3 and photolyzed ci-IP3 to enable independent activation of GPCRs and delivery of i-IP3 to IP3Rs. Activation of H1 receptors in HeLa cells or M3 muscarinic receptors in HEK293 cells in the presence of IP3KC reduced the frequency of Ca2+ puffs evoked exogenously by photolysis of ci-IP3 without affecting puff amplitude or kinetics. This inhibition was entirely mediated by PI(4,5)P2 depletion. PI(4,5)P2 depletion significantly reduced the likelihood of Ca2+ puffs progressing to a global Ca2+ signal, but once the transition threshold was reached, the amplitude of the global signal was indistinguishable in the presence and absence of PI(4,5)P2. PI(4,5)P2 depletion did not affect the subcellular distribution of IP3Rs. I suggest that PI(4,5)P2 primes IP3Rs to respond to IP3 by partially occupying the receptor’s IP3-binding site. Increasing PI(4,5)P2 levels in the PM did not further activate IP3Rs, suggesting that basal PI(4,5)P2 achieves the maximal effect. It is unclear whether this occurs with all IP3-binding sites occupied by PI(4,5)P2 or whether physical barriers constrain the number of sites that can be occupied. My results establish that PI(4,5)P2 primes IP3Rs to respond, and that as GPCRs stimulate IP3 formation they also deplete PI(4,5)P2, relieving this priming stimulus and resetting IP3R sensitivity. Dual regulation of IP3Rs by PI(4,5)P2 and IP3 through GPCRs thus controls the transition from local to global Ca2+ signals."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["b81a61e3d1494904f49e0114f1d5b495","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["Dual regulation of inositol 1,4,5-trisphosphate receptors by inositol 1,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate"]}]}],"canonical_facts":{"dc:contributor.advisor":["Ladds, Graham"],"dc:creator":["Ivanova, Adelina Adelin"],"dc:date.issued":["2024-06-08"],"dc:description.abstract":["Ca2+ is a universal and effective intracellular messenger, which holds a central role in the regulation of a vast array of cellular processes. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are key signal integrators, which transform extracellular stimuli into intracellular Ca2+ signals. Only immobilised IP3Rs, licensed by association with KRas-induced actin-interacting protein (KRAP), can respond through IP3-mediated Ca2+ release. These licensed IP3Rs are tethered on actin near membrane contact sites (MCS) between the endoplasmic reticulum (ER) and plasma membrane (PM), where store-operated Ca2+ entry (SOCE) takes place. Uncovering the mechanisms that govern IP3R regulation is an essential element in understanding the spatial and temporal patterns of Ca2+ signalling. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor, but functionally diverse lipid at the PM. In the canonical Ca2+ signalling cascade, activation of PM-resident receptors such as G-protein coupled receptors (GPCRs) causes phospholipase C (PLC) to hydrolyse PI(4,5)P2, producing inositol 1,4,5-trisphosphate (IP3) which diffuses through the cytoplasm to IP3Rs, enabling Ca2+ release from the ER. The stimulus intensity governs the amount of IP3 produced and in consequence, the extent of IP3R activation, starting from brief, localised Ca2+ puffs, and progressing to cell-wide global Ca2+ waves. Tight control of the transition from local to global Ca2+ signals is central to the downstream consequences of receptor activation. PI(4,5)P2, with its essential roles in F-actin nucleation and formation of the SOCE complex at ER-PM MCS, is thus a potential regulator of IP3R activity in addition to its role in providing IP3. Towards exploring possible additional roles of PI(4,5)P2 in regulating Ca2+ signalling via IP3Rs, I have assessed three strategies for selective depletion of PI(4,5)P2 at the PM: pharmacological inhibition of the synthesis of phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2, gene silencing of the 5-kinases that convert PI(4)P to PI(4,5)P2, and expression of a rapamycin-inducible heterodimerization system that allows translocation of a PI(4,5)P2-specific 5-phosphatase (herein referred to as the ‘’5-PTASE system’’) to the PM. Pharmacological or siRNA-mediated inhibition of the relevant kinases in the PI(4,5)P2 metabolic cycle did not successfully attenuate Ca2+ signals in response to histamine in HeLa cells, suggesting that PLC-sensitive PI(4,5)P2 pools remain available after treatment. On the contrary, I have shown that the 5-PTASE system caused global depletion of PI(4,5)P2 at the PM using a genetically encoded, PI(4,5)P2-selective fluorescent sensor, and showed that Ca2+ signals in response to histamine in HeLa cells were attenuated. The method thereby allows acute and near-complete depletion of PM-associated PI(4,5)P2. Using the validated 5-PTASE system for PM PI(4,5)P2 depletion, and uniform delivery of i-IP3 to the cytosol via uncaging of the exogenously supplied photolabile caged ci-IP3, revealed that PI(4,5)P2 depletion significantly reduced the frequency of Ca2+ puffs in HeLa and HEK293 cells without affecting puff amplitude or kinetics. PI(4,5)P2 regulation was confirmed to extend to all three IP3R subtypes. As PI(4,5)P2 depletion may lead to reduction of basal IP3 levels, I employed two complementary approaches to assess whether a loss of basal IP3 is responsible for the reduced Ca2+ puff frequency. Reducing basal IP3 levels by inhibiting PLC activity with U73122 or by overexpressing cytosolic IP3 kinase C (IP3KC) did not reduce the frequency of Ca2+ puffs evoked by photolysis of ci-IP3. I conclude that PI(4,5)P2 regulates IP3R activity in parallel to providing IP3. As PI(4,5)P2 levels at the PM are dynamically controlled during signalling when PI(4,5)P2 is consumed to produce IP3, I developed methods to uncouple stimulation of GPCRs that evoke IP3 formation from delivery of IP3 to IP3Rs while retaining opportunities to stimulate IP3Rs directly. I expressed IP3KC to intercept endogenous IP3 and photolyzed ci-IP3 to enable independent activation of GPCRs and delivery of i-IP3 to IP3Rs. Activation of H1 receptors in HeLa cells or M3 muscarinic receptors in HEK293 cells in the presence of IP3KC reduced the frequency of Ca2+ puffs evoked exogenously by photolysis of ci-IP3 without affecting puff amplitude or kinetics. This inhibition was entirely mediated by PI(4,5)P2 depletion. PI(4,5)P2 depletion significantly reduced the likelihood of Ca2+ puffs progressing to a global Ca2+ signal, but once the transition threshold was reached, the amplitude of the global signal was indistinguishable in the presence and absence of PI(4,5)P2. PI(4,5)P2 depletion did not affect the subcellular distribution of IP3Rs. I suggest that PI(4,5)P2 primes IP3Rs to respond to IP3 by partially occupying the receptor’s IP3-binding site. Increasing PI(4,5)P2 levels in the PM did not further activate IP3Rs, suggesting that basal PI(4,5)P2 achieves the maximal effect. It is unclear whether this occurs with all IP3-binding sites occupied by PI(4,5)P2 or whether physical barriers constrain the number of sites that can be occupied. My results establish that PI(4,5)P2 primes IP3Rs to respond, and that as GPCRs stimulate IP3 formation they also deplete PI(4,5)P2, relieving this priming stimulus and resetting IP3R sensitivity. Dual regulation of IP3Rs by PI(4,5)P2 and IP3 through GPCRs thus controls the transition from local to global Ca2+ signals."],"dc:format.checksum.md5":["b81a61e3d1494904f49e0114f1d5b495","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.112415"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/37edb92f-4a98-4b67-8e41-fb1da0cb17e5/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/374317"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/85d0f03d-ef0e-4e8e-b460-802ab1fc510c/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["Ca2+","endoplasmic reticulum","IP3R","PI(4,5)P2","rapamycin","signalling"],"dc:title":["Dual regulation of inositol 1,4,5-trisphosphate receptors by inositol 1,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:24Z"}