Abstract：
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.
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.
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.
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.
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.
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.
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.
摘要：
目的：肌醇1,4,5-三磷酸受体（IP3Rs）是细胞内Ca2+释放通道，在细胞功能中起着至关重要的作用。目前的IP3R抑制剂遭受脱靶效应和对三种不同IP3R亚型的差的选择性。我们开发了一种新型的IP3Rs肽抑制剂，并确定了其对连接蛋白-43（Cx43）半通道的影响，它们被IP3R刺激共激活。
方法：IP3RPEP6是通过计算机分子对接研究开发的，并通过IP3R2通道和卡巴胆碱诱导的IP3介导的Ca2反应的核上膜片钳实验表征IP3R1，2或3表达细胞，三IP3RKO细胞和星形胶质细胞。通过膜片钳和ATP释放方法研究了Cx43半通道，和通过用Gap19肽抑制。IP3RPEP6与IP3R的相互作用通过共免疫沉淀和亲和力下拉测定来验证。
结果：IP3RPEP6浓度依赖性地降低了IP3R2通道的开放概率，并以IP3R2（〜3.9μM）结论：IP3RPEP6抑制IP3R2/R3的浓度对IP3R1的影响有限。IP3R激活触发半通道打开，强烈影响IP3触发的Ca2响应的幅度和浓度依赖性。
方法：IP3RPEP6是通过计算机分子对接研究开发的，并通过IP3R2通道和卡巴胆碱诱导的IP3介导的Ca2反应的核上膜片钳实验表征IP3R1，2或3表达细胞，三IP3RKO细胞和星形胶质细胞。通过膜片钳和ATP释放方法研究了Cx43半通道，和通过用Gap19肽抑制。IP3RPEP6与IP3R的相互作用通过共免疫沉淀和亲和力下拉测定来验证。
结果：IP3RPEP6浓度依赖性地降低了IP3R2通道的开放概率，并以IP3R2（〜3.9μM）
