SK3 channel trafficking in endothelial cells

项目摘要 内皮细胞排列在所有血管的壁上，调节多种功能，包括收缩功能。 和全身血压。内皮细胞功能障碍是几种心血管疾病的标志，但 涉及的病理机制尚不清楚。内皮细胞表达小电导钙激活 钾(SK3)通道，调节收缩能力。内皮细胞SK3通道产生的电流(I) 信元是信道数(N)、它们的开放概率(PO)和幅度(I)的乘积，使得 I=N.PO.i。以前的研究主要集中在识别调节表面SK活性的机制。 内皮细胞中的通道。相比之下，控制表面SK3通道的数量(N)的机制 人们对血管内皮细胞知之甚少。重要的是，目前尚不清楚生理刺激是否调节 丰富的表面SK3通道可以改变动脉的收缩能力。同样不确定的是，病理改变是否 在对照的体表SK3通道中，高血压期间发生丰富，导致衰减 血管扩张。使用多种方法，我们提供了血管扩张剂刺激激活的证据 迅速增加内皮细胞表面SK3通道丰度的转运机制 血管扩张。初步数据还表明，在高血压期间，SK3通道运输功能障碍， 它减弱了这种血管扩张信号机制。在这项提案中，我们将调查三个具体的 目标。目标1将研究生理刺激改变表面丰度的信号机制 并检测它们的功能意义。目标2将审查贩运机制， 控制面SK3通道在内皮细胞中调节动脉的收缩能力。目标3将研究这一假设 高血压与血管内皮细胞SK3通道运输的病理改变有关 通过这些蛋白质抑制血管扩张。使用的方法包括生物素化、Western blotting、FRET、RNAi、 共激发态，免疫荧光，超分辨显微镜，膜片钳电生理，膜电位 记录、细胞内钙成像、动脉肌图、血压遥测和转基因小鼠。这 该项目将提供关于SK3通道贩运的血管调节的重要新信息 内皮细胞。

Project Summary Endothelial cells line the wall of all blood vessels and regulate a wide variety of functions, including contractility and systemic blood pressure. Endothelial cell dysfunction is a hallmark of several cardiovascular diseases, but pathological mechanisms involved are unclear. Endothelial cells express small-conductance calcium-activated potassium (SK3) channels, which regulate contractility. Currents (I) generated by SK3 channels in endothelial cells is the product of the number of channels (N), their open probability (PO) and amplitude (i), such that I=N.PO.i. Previous studies have focused on identifying mechanisms that regulate the activity of surface SK channels in endothelial cells. In contrast, mechanisms that control the number (N) of surface SK3 channels in endothelial cells are poorly understood. Importantly, it is unclear whether physiological stimuli regulate the abundance of surface SK3 channels to alter arterial contractility. Similarly uncertain is if pathological alterations in the control of surface SK3 channel abundance occurs during hypertension, leading to attenuated vasodilation. Using a wide variety of approaches, we provide evidence that vasodilator stimuli activate trafficking mechanisms that rapidly increase surface SK3 channel abundance in endothelial cells to induce vasodilation. Preliminary data also suggest that SK3 channel trafficking is dysfunctional during hypertension, which attenuates this vasodilatory signaling mechanism. In this proposal, we will investigate three specific aims. Aim 1 will investigate signaling mechanisms by which physiological stimuli alter the surface abundance of SK3 channels and examine their functional significance. Aim 2 will examine trafficking mechanisms that control surface SK3 channels in endothelial cells to modify arterial contractility. Aim 3 will study the hypothesis that hypertension is associated with pathological alterations in SK3 channel trafficking in endothelial cells that inhibit vasodilation by these proteins. Methods used will include biotinylation, Western blotting, FRET, RNAi, co-IP, immunofluorescence, super-resolution microscopy, patch-clamp electrophysiology, membrane potential recording, intracellular Ca2+ imaging, arterial myography, blood pressure telemetry and transgenic mice. This project will provide significant novel information concerning vasoregulation by SK3 channel trafficking in endothelial cells.