IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels

Abstract

Aim: Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+-release channels with crucial roles in cell function. Current IP3R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3R subtypes. We developed a novel peptide inhibitor of IP3Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3R stimulation. Methods: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3R2 channels and carbachol-induced IP3-mediated Ca2+ responses in IP3R1, 2 or 3 expressing cells, triple IP3R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3Rs were verified by co-immunoprecipitation and affinity pull-down assays. Results: IP3RPEP6 concentration-dependently reduced the open probability of IP3R2 channels and competitively inhibited IP3Rs in an IC50 order of IP3R2 (~3.9 μM) < IP3R3 (~4.3 μM)