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

AIM

Inositol 1,4,5-trisphosphate receptors (IP Rs) are intracellular Ca -release channels with crucial roles in cell function. Current IP R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP R subtypes. We developed a novel peptide inhibitor of IP Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP R stimulation.

METHODS

IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP R2 channels and carbachol-induced IP -mediated Ca responses in IP R1, 2 or 3 expressing cells, triple IP R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP Rs were verified by co-immunoprecipitation and affinity pull-down assays.

RESULTS

IP3RPEP6 concentration-dependently reduced the open probability of IP R2 channels and competitively inhibited IP Rs in an IC order of IP R2 (3.9 μM) < IP R3 (4.3 μM) < IP R1 (~9.0 μM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP R2 but not with IP R1; interaction with IP R3 varied between cell types. The IC of IP3RPEP6 inhibition of carbachol-induced Ca responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP -Ca responses and strongly increased the EC of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP R-inhibiting peptide.

CONCLUSION

IP3RPEP6 inhibits IP R2/R3 at concentrations that have limited effects on IP R1. IP R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP -triggered Ca responses.