Apical localization of ITPK1 enhances its ability to be a modifier gene product in a murine tracheal cell model of cystic fibrosis

A new aspect of research into the pathogenesis of cystic fibrosis (CF) is a genetics-based search for 'modifier genes' that may affect the severity of CF lung disease. Using an alternative, cell biological approach, we show that ITPK1 should be considered a modifier gene. ITPK1 synthesizes an intracellular signal, inositol (3,4,5,6)-tetrakisphosphate [Ins(3,4,5,6)P₄]. A bio-activatable, cell-permeable analogue of Ins(3,4,5,6)P₄ inhibited Ca²⁺-dependent secretion of Cl^- from polarized monolayers of immortalized mouse tracheal epithelial cells (MTEs). Analysis by high-pressure liquid chromatography showed endogenous Ins(3,4,5,6)P₄ levels in CF MTEs were approximately 60% below those in wild-type MTEs (P<0.03). This adaptation, which improves purinergic activation of Ca²⁺-dependent Cl^- secretion in CF MTEs, was exceptionally specific; there was no effect upon the cellular levels of all the other inositol phosphate signals. Real-time PCR provided the explanation: the level of ITPK1 expression in wild-type MTEs was twice as high as that in CF MTEs (P<0.002). The biological impact of this differential gene expression is amplified by ITPK1 being concentrated at the apical membrane of MTEs, which we discovered following confocal immunofluorescence microscopy. Compartmentalization of Ins(3,4,5,6)P₄ synthesis adjacent to its site of action will enhance its regulatory capacity.