Inositol hexakisphosphate kinase 1 regulates polyphosphate synthesis by maintaining V-ATPase-dependent secretory...

Polyphosphate (polyP), a linear polymer of orthophosphate residues, is enriched in secretory granules in specialised mammalian cell types including platelets and mast cells. Although polyP released during activation and degranulation of these cells has been shown to promote blood clotting and inflammation, little is known about the mechanisms governing polyP synthesis in these granules. In mice, the loss of IP6K1, an enzyme that catalyses the production of 5-InsP₇, has been shown to result in depletion of platelet polyP and impaired hemostasis. Here, we use the rat mast cell line RBL-2H3 as a model to study the regulation of polyP synthesis in secretory granules. By monitoring real-time polyP synthesis in isolated mast cell granules, we demonstrate that ATP is the substrate fuelling granule polyP production. By the use of inhibitors, we show that accumulation of polyP in granules requires an intact transmembrane proton gradient maintained by vacuolar H⁺ATPase (V-ATPase). In RBL-2H3 cells, depletion of IP6K1 led to a substantial reduction in cellular polyP levels and defective accumulation of polyP, serotonin, and tryptase inside granules. Cells with reduced IP6K1 showed a profound loss of granule acidification, correlating with downregulated levels of V₁ subunits of V-ATPase. Adding back active or catalytically inactive IP6K1 rescued the expression of V-ATPase V₁ subunits, reversed granule deacidification, and restored polyP levels in IP6K1-depleted cells. Together, these data unveil a role for IP6K1 in maintaining granule pH and thereby supporting polyP synthesis in mammals.