Photolysis of Caged Inositol Pyrophosphate InsP₈ Directly Modulates Intracellular Ca²⁺ Oscillations and Controls C2AB Domain Localization

Abstract

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP 5 ) has greatly improved, the functions of the inositol octaphosphates ((PP) 2 -InsP 4 ) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP) 2 -InsP 4 derivatives and apply them to study the functions in living β-cells. Photorelease of the naturally occurring isomer 1,5-(PP) 2 -InsP 4 led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP) 2 -InsP 4 , 5-PP-InsP 5 , 1-PP-InsP 5 , 3-PP-InsP 5 ) showed no immediate effect. Furthermore, uncaging of 1,5-(PP) 2 -InsP 4 but not 3,5-(PP) 2 -InsP 4 induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP) 2 -InsP 4 impacts β-cell activity by regulating granule localization and/or priming and calcium signaling in concert.

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

“…Inositol pyrophosphates ubiquitously occur in eukaryotic cells 4 and serve as metabolic signalling molecules critically involved in e.g. phosphate homeostasis 5,6 , insulin sensitivity 7 , calcium oscillations 8,9 , vesicular trafficking 10 , apoptosis 11 , DNA repair 12 , immune signalling 13 , and others. The plethora of important processes under the control of inositol pyrophosphates calls for a deeper understanding of their cellular abundance, fluctuation, and localisation.…”

Metabolism and Functions of Inositol Pyrophosphates: Insights Gained from the Application of Synthetic Analogues

“…Subsequently, UV-mediated uncaging of 5-InsP 7 was found to block Ca 2+ oscillations in the mouse pancreatic insulinoma (MIN6) cell-line, after a delay of 15 min [ 70 ]. In a subsequent study using caged 1,5-InsP 8 , its photorelease instantly stopped Ca 2+ oscillations [ 71 ]. Thus, in the earlier study, it seems likely that the delay was due to 5-InsP 7 phosphorylation to 1,5-InsP 8 .…”

“…On the other hand, Jessen and colleagues also described an apparently separate effect of 1,5-InsP 8, which, theoretically at least, could promote exocytosis, through PP-InsP’s interaction with synaptotagmin-like protein 4 (Slp-4)/granuphilin [ 71 ]. The latter is required for insulin granules to dock to the plasma membrane, but such vesicles are not fusion-competent until granuphilin is subsequently detached [ 73 ].…”

“…A mechanism for granuphilin release has previously been proposed: competition from cytosolic InsP 6 for plasma-membrane associated PtdIns(4,5P) 2 binding to granuphilin’s C2A and C2B binding domains [ 74 ]. However, 1,5-InsP 8 was shown in Jessen’s study to also bind to each of these two domains in vitro (K d = 0.6 and 5 µM, respectively [ 71 ], as compared to 1.8 and 21 µM for InsP 6 [ 74 ]). Moreover, the caged 1,5-InsP 8 proved useful in validating PP-InsP’s interaction with granuphilin in intact cells; a transfected and membrane-associated eGFP-tagged C2AB domain from granuphilin was found to redistribute into the cytoplasm following uncaging of the 1,5-InsP 8 [ 71 ].…”

“…However, 1,5-InsP 8 was shown in Jessen’s study to also bind to each of these two domains in vitro (K d = 0.6 and 5 µM, respectively [ 71 ], as compared to 1.8 and 21 µM for InsP 6 [ 74 ]). Moreover, the caged 1,5-InsP 8 proved useful in validating PP-InsP’s interaction with granuphilin in intact cells; a transfected and membrane-associated eGFP-tagged C2AB domain from granuphilin was found to redistribute into the cytoplasm following uncaging of the 1,5-InsP 8 [ 71 ]. That is, 1,5-InsP 8 was shown to be functionally active even in the presence of steady-state levels of InsP 6 .…”