Analyzing the role of the putative inositol 1,3,4,5-tetrakisphosphate receptor GAP1IP4BP in intracellular Ca2+ homeostasis

Abstract

Inositol 1,3,4,5-tetrakisphosphate (IP4) has been linked to a potential role in the regulation of intracellular free Ca2+ concentration ([Ca2+]i) following cellular stimulation with agonists that activate phosphoinositide-specific phospholipase C. However, despite many studies, the function of IP4 remains unclear and indeed there is still some debate over whether it has a function at all. Here we have used various molecular approaches to address whether manipulation of the potential IP4 receptor, GAP1IP4BP, affects [Ca2+]i following cellular stimulation. Using single cell imaging, we show that the overexpression of a constitutively active and a potential dominant negative form of GAP1IP4BP appear to have no effect on Ca2+ mobilization or Ca2+ entry following stimulation of HeLa cells with histamine. In addition, through the use of small interfering RNA duplexes, we have examined the effect of suppressing endogenous GAP1IP4BP production on [Ca2+]i. In HeLa cells in which the endogenous level of GAP1IP4BP has been suppressed by ∼95%, we failed to observe any effect on Ca2+ mobilization or Ca2+ entry following histamine stimulation. Thus, using various approaches to manipulate the function of endogenous GAP1IP4BP in intact HeLa cells, we have been unable to observe any detectable effect of GAP1IP4BP on [Ca2+]i.