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Dual regulation of inositol 1,4,5-trisphosphate receptors by inositol 1,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate https://doi.org/10.17863/CAM.112415 Ivanova, Adelina Adelin Ladds, Graham 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. University of Cambridge 2024-06-08 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/374317 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/37edb92f-4a98-4b67-8e41-fb1da0cb17e5/download b81a61e3d1494904f49e0114f1d5b495 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/85d0f03d-ef0e-4e8e-b460-802ab1fc510c/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Ca2+ endoplasmic reticulum IP3R PI(4,5)P2 rapamycin signalling