Protein kinase A-phosphorylated KV1 channels in PSD95 signaling complex contribute to the resting membrane potential and diameter of cerebral arteries.
Protein kinase A-phosphorylated KV1 channels in PSD95 signaling complex contribute to the resting membrane potential and diameter of cerebral arteries.
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DOI：
10.1161/circresaha.114.303167
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发表时间：
2014-04-11
影响因子：
20.1
通讯作者：
Rhee SW
中科院分区：
文献类型：
--
作者：
Moore CL;Nelson PL;Parelkar NK;Rusch NJ;Rhee SW
突触后密度-95（Postsynaptic density-95，PSD 95）是一种支架蛋白，与电压门控的Shaker型K+（KV 1）通道相关，并促进脑循环血管平滑肌细胞（cVSMC）中KV 1通道的表达。然而，PSD 95在介导cVSMC中的分子信号传导中的生理作用是未知的。我们探索了PSD 95和KV 1通道之间的特异性相互作用是否能够使cVSMC中KV 1通道的PKA磷酸化以促进血管舒张。设计了一种与KV1.2α C端PDZ结合基序对应的膜渗透性肽（KV 1-C肽），作为显性负性肽，以破坏KV 1通道与PSD 95的结合。将KV 1-C肽应用于插管加压CA快速诱导血管收缩和去极化cVSMC。这些事件对应于PSD 95和KV 1蛋白的免疫共沉淀减少而不改变表面表达。通过颅窗原位成像的大脑中小动脉也对局部应用KV 1-C肽作出快速收缩反应。膜片钳记录证实KV 1-C肽减弱cVSMCs中KV 1通道阻断剂（Psora 4）敏感性电流。使用磷酸化PKA底物抗体的Western印迹显示，暴露于KV 1-C肽的CA显示KV1.2α亚基的磷酸化明显减少。最后，磷酸酶抑制剂减弱了KV 1-C肽介导的和PKA抑制肽介导的血管收缩。这些研究结果提供了初步证据，PKA磷酸化的KV 1通道是通过与PSD 95在CA中的动态关联，并表明，这种关联的破坏可能会损害脑血管舒张和血流。
Postsynaptic density-95 (PSD95) is a scaffolding protein that associates with voltage-gated, Shaker-type K+ (KV1) channels and promotes the expression of KV1 channels in vascular smooth muscle cells (cVSMCs) of the cerebral circulation. However, the physiological role of PSD95 in mediating molecular signaling in cVSMCs is unknown. We explored whether a specific interaction between PSD95 and KV1 channels enables PKA phosphorylation of KV1 channels in cVSMCs to promote vasodilation Rat cerebral arteries (CA) were used for analyses. A membrane-permeable peptide (KV1-C peptide) corresponding to the PDZ binding motif in the C-terminus of KV1.2α was designed as a dominant negative peptide to disrupt the association of KV1 channels with PSD95. Application of KV1-C peptide to cannulated, pressurized CA rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced co-immunoprecipitation of the PSD95 and KV1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of KV1-C peptide. Patch-clamp recordings confirmed that KV1-C peptide attenuates KV1 channel blocker (Psora4)-sensitive current in cVSMCs. Western blots employing a phospho-PKA substrate antibody revealed CA exposed to KV1-C peptide showed markedly less phosphorylation of KV1.2α subunits. Finally, phosphatase inhibitors blunted both KV1-C peptide-mediated and PKA inhibitor peptide-mediated vasoconstriction. These findings provide initial evidence that PKA phosphorylation of KV1 channels is enabled by a dynamic association with PSD95 in CA, and suggest that a disruption of such association may compromise cerebral vasodilation and blood flow.