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

Aim: Inositol 1,4,5-trisphosphate receptors (IP₃ Rs) are intracellular CA²⁺ -release channels with crucial roles in cell function. Current IP₃ R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP₃ R subtypes. We developed a novel peptide inhibitor of IP₃ Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP₃ R stimulation.

Methods: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP₃ R2 channels and carbachol-induced IP₃ -mediated CA²⁺ responses in IP₃ R1, 2 or 3 expressing cells, triple IP₃ 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 IP₃ Rs were verified by co-immunoprecipitation and affinity pull-down assays.

Results: IP3RPEP6 concentration-dependently reduced the open probability of IP₃ R2 channels and competitively inhibited IP₃ Rs in an IC₅₀ order of IP₃ R2 (~3.9 μM) < IP₃ R3 (~4.3 μM) < IP₃ R1 (~9.0 μM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP₃ R2 but not with IP₃ R1; interaction with IP₃ R3 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 IP₃ R-inhibiting peptide.

Conclusion: IP3RPEP6 inhibits IP₃ R2/R3 at concentrations that have limited effects on IP₃ R1. IP₃ R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP₃ -triggered CA²⁺ responses.

Ca2+ signaling; IP3; astrocytes; connexin-43; hemichannel; inositol 1,4,5-trisphosphate; peptide inhibitor.