Investigation of the Inhibitory Effects of the Benzodiazepine Derivative, 5-BDBD on P2X4 Purinergic Receptors by two Complementary Methods

Abstract

Background/Aims: ATP-gated P2X₄ purinergic receptors (P2X₄Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X₄Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X₄Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X₄Rs. Methods: We investigated ATP-induced intracellular Ca²⁺ signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X₄Rs. Results: Our data show that ATP (< 1 μM) stimulates P2X₄R-mediated Ca²⁺ influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca²⁺ signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X₄Rs. Conclusions: Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X₄Rs although their mechanisms of actions are different.