The Epac1 Signaling Pathway Regulates Cl- Secretion via Modulation of Apical KCNN4c Channels in Diarrhea
The Epac1 Signaling Pathway Regulates Cl- Secretion via Modulation of Apical KCNN4c Channels in Diarrhea
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DOI：
10.1074/jbc.m113.467860
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发表时间：
2013-07-12
影响因子：
4.8
通讯作者：
Hoque, Kazi Mirajul
中科院分区：
文献类型：
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作者：
Sheikh, Irshad Ali;Koley, Hemanta;Hoque, Kazi Mirajul
肠上皮细胞顶膜表达中电导K+通道(KCNN4)，为氯离子的分泌提供动力。然而，它在腹泻中的作用以及Epac1的调节作用尚不清楚。以前我们已经证实，Epac1与cAMP结合后，通过刺激Rap2-磷脂酶C epsilon-[Ca~(2+)](I)信号，激活一种不依赖PKA的氯离子分泌机制。在此，我们报道了Epac1通过其下游的Rap1a-RhoA-Rho相关蛋白激酶(ROCK)信号通路调节KCNN4c通道的表面表达，以维持持续的氯离子分泌。去除Epac1蛋白和顶端加入特异性KCNN4抑制剂TRAM-34可显著抑制cAMP刺激的T84WT细胞的Cl-分泌和顶端K+电导(I-K(AP))。Epac1激动剂8-(4-氯苯硫基)-2‘-O-甲基腺苷3’，5‘-环磷酸腺苷作用于T84WT细胞后的电流-电压关系显示，在T84WT细胞中存在内向整流和TRAM-34敏感的K+通道，而Epac1KDT84细胞中没有这种通道。在Epac1KDT84细胞中重建的共聚焦图像显示KCNN4c蛋白重新分布到根尖下的细胞内，生物素化实验显示与T84WT细胞相比，KCNN4c蛋白的表面表达降低了83%。进一步的研究表明，Epac1激动剂激活Rap1以促进I-K(AP)。RhoA抑制剂(GGTI298)和ROCK抑制剂(H1152)均显著降低cAMP激动剂刺激的I-K(AP)，而后者还减少了KCNN4c与顶膜标记小麦胚凝集素在T84WT细胞中的共存。在体小鼠回肠循环实验显示，当TRAM-34、GGTI298或H1152与霍乱毒素一起注射到回肠循环中时，液体聚集减少。我们的结论是，RhoA-ROCK参与的Epac1的Rap1a依赖信号是通过调节顶端KCNN4c通道来调节肠液转运的重要调节因子，这一发现在腹泻疾病中具有潜在的治疗价值。
The apical membrane of intestinal epithelia expresses intermediate conductance K+ channel (KCNN4), which provides the driving force for Cl- secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl- secretion via stimulation of Rap2-phospholipase C epsilon-[Ca2+](i) signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl- secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl- secretion and apical K+ conductance (I-K(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2'-O- methyladenosine 3',5'-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K+ channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed similar to 83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate I-K(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated I-K(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.