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

Abstract

AimInositol 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.MethodsIP3RPEP6 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.ResultsIP3RPEP6 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) < IP3R1 (~9.0 μM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co‐immunoprecipitated with IP3R2 but not with IP3R1; interaction with IP3R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol‐induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19‐inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3‐Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl‐8G‐IP3RPEP6 as a membrane‐permeable IP3RPEP6 version allowing extracellular application of the IP3R‐inhibiting peptide.ConclusionIP3RPEP6 inhibits IP3R2/R3 at concentrations that have limited effects on IP3R1. IP3R activation triggers hemichannel opening, which strongly affects the amplitude and concentration‐dependence of IP3‐triggered Ca2+ responses.

Calcium Regulation of Connexin Hemichannels

“…Indeed, in connexin isoforms such as Cx32 and Cx43, the voltage alone can trigger HC opening only at unphysiological values ≥ +40 mV [ 59 , 72 , 74 , 107 ]. Instead, a physiological [Ca 2+ ] c rise alone triggered by InsP 3 can stimulate HC opening [ 74 , 155 ] while dramatically increasing the voltage sensitivity [ 107 ].…”