PI3 kinase inhibitor PI828 uncouples aminergic GPCRs and Ca2+ mobilization irrespectively of its primary target

Abstract

The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied Ca²⁺ signaling initiated by aminergic agonists in a variety of different cells and analyzed effects of the PI3K inhibitor PI828 on cell responsiveness. It turned out that PI828 inhibited Ca²⁺ transients elicited by acetylcholine (ACh), histamine, and serotonin, but did not affect Ca²⁺ responses to norepinephrine and ATP. Another PI3K inhibitor wortmannin negligibly affected Ca²⁺ signaling initiated by any one of the tested agonists. Using the genetically encoded PIP₃ sensor PH(Akt)-Venus, we confirmed that both PI828 and wortmannin effectively inhibited PI3K and ascertained that this kinase negligibly contributed to ACh transduction. These findings suggested that PI828 inhibited Ca²⁺ responses to aminergic agonists tested, involving an unknown cellular mechanism unrelated to the PI3K inhibition. Complementary physiological experiments provided evidence that PI828 could inhibit Ca²⁺ signals induced by certain agonists, by acting extracellularly, presumably, through their surface receptors. For the muscarinic M3 receptor, this possibility was verified with molecular docking and molecular dynamics. As demonstrated with these tools, wortmannin could be bound in the extracellular vestibule at the muscarinic M3 receptor but this did not preclude binding of ACh to the M3 receptor followed by its activation. In contrast, PI828 could sterically block the passage of ACh into the allosteric site, preventing activation of the muscarinic M3 receptor.