Coronary Artery Regulation by Small Conduction Ca2+-activated K+ Channels

描述（由申请人提供）：内皮功能丧失是冠状动脉疾病（CAD）发展的关键因素。然而，内皮依赖性信号传导，特别是负责冠状动脉张力的内皮控制的潜在生理机制知之甚少。我们最近已经证明，小电导钙激活的K+通道，SK 3，是在外周动脉内皮功能的基本控制器，促进持续扩张成比例的表达水平。我们在冠状动脉中的初步数据表明，在生理压力和流量下，组成性SK 3通道活动对抗稳态张力。我们现在已经确定了冠状动脉内皮细胞中产生的重复自发Ca 2+瞬变，这可能为SK 3通道激活提供基本刺激。特别是，这些Ca 2+信号的接近孔内弹性膜（IEL）以及SK 3通道和特定的间隙连接连接蛋白表达沿着内皮血管平滑肌（VSM）接口，建议存在一个本地化的肌内皮信号复合物。我们假设在冠状动脉生理条件下，膜SK 3通道的重复Ca 2+依赖性激活驱动紧张性内皮超极化，其通过IEL孔通过间隙连接快速传递到邻近平滑肌。此外，我们认为雌激素诱导的内皮SK 3通道表达上调放大了这种内皮源性超极化（EDH）。为了充分解决这一假设，我们制定了两个具体目标。目的1将直接评估自发性Ca 2+事件与完整冠状动脉内皮中SK 3依赖性膜电位超极化的功能偶联，以及这种效应是否因差异SK 3表达（即通过直接遗传操作或雌激素）和剪切应力而改变。目的2将评估IEL孔部位的局灶性Ca 2+依赖性SK 3激活是否允许内膜下VSM通过间隙连接直接超极化，从而降低VSM Ca 2+并促进冠状动脉扩张。为了实现这些目标，我们将采用最先进的创新方法，包括1）在完整冠状动脉中同时进行高速共聚焦成像和细胞内电生理测量，2）独特的遗传小鼠模型（SK 3 T/T），其中可以通过实验控制SK 3表达，以明确识别SK 3通道的具体影响，和3）靶向特异性间隙连接连接蛋白的新型肽抑制剂。这项工作将提供一个基本的内皮信号和生理血管调节冠状动脉的范例，并确定潜在的细胞靶点，为未来的治疗对CAD。

DESCRIPTION (provided by applicant): Loss of endothelial function is a critical factor in the development of coronary artery disease (CAD). Still, endothelium dependent signaling and particularly the underlying physiological mechanism responsible for endothelial control of coronary artery tone are poorly understood. We have recently demonstrated that small conductance Ca2+-activated K+ channels, SK3, are fundamental controllers of endothelial function in peripheral arteries, promoting sustained dilation proportional to their level of expression. Our preliminary data in coronary arteries indicate that under physiological pressure and flow, constitutive SK3 channel activity opposes steady-state tone. We have now identified repetitive spontaneous Ca2+ transients generated in coronary endothelial cells that may provide the fundamental stimulus for SK3 channel activation. In particular, the proximity of these Ca2+ signals to holes in the internal elastic lamina (IEL) as well as to SK3 channels and specific gap junction connexins expressed along the endothelial-vascular smooth muscle (VSM) interface, suggest the existence of a localized myoendothelial signaling complex. We hypothesize that under physiological conditions in coronary arteries, repeated Ca2+-dependent activation of membrane SK3 channels drives tonic endothelial hyperpolarization, which is rapidly communicated through IEL holes to adjacent smooth muscle through gap junctions. Moreover, we propose that estrogen-induced upregulation of endothelial SK3 channel expression amplifies this endothelium-derived hyperpolarization (EDH). To fully address this hypothesis, we have formulated two specific aims. Aim 1 will directly assess the functional coupling of spontaneous Ca2+ events to SK3-dependent membrane potential hyperpolarization in the endothelium of intact coronary arteries and whether this effect is altered by differential SK3 expression (i.e. via direct genetic manipulation or estrogen) and shear stress. Aim 2 will assess whether focal Ca2+-dependent SK3 activation at sites of IEL holes allows for direct hyperpolarization of subintimal VSM via gap junctions, thereby reducing VSM Ca2+ and promoting coronary artery dilation. In pursuit of these aims, we will apply state-of-the-art and innovative approaches including 1) simultaneous high-speed confocal imaging and intracellular electrophysiological measurements in intact coronary arteries, 2) a unique genetic mouse model (SK3T/T) in which SK3 expression can be experimentally controlled to unequivocally discern the specific impact of SK3 channels, and 3) novel peptide inhibitors to target specific gap junction connexins. This work will provide a paradigm of fundamental endothelial signaling and physiological vasoregulation in coronary arteries, and identify potential cellular targets for future therapies against CAD.