Tuning L-Type Ca Channel Activity in Arterial Smooth Muscle by Kv Channel-Mediated Clustering

项目摘要 二氢吡啶敏感、L型Cav1.2和延迟整流Kv2.1通道在 动脉平滑肌兴奋性和收缩的调节。Cav1.2通道的一个显著特点是 它们形成集群，在其中它们经历动态的、相互的相互作用，从而允许功能耦合 相邻通道，从而放大钙信号，这对肌源性疾病的发展至关重要 语气。然而，目前控制Cav1.2聚集的机制尚不清楚。修剪机和 桑塔纳实验室已联手解决这一根本问题。来自我们实验室的新的初步数据 提出一个新的模型，代表相对于普遍接受的规范角色的范式转变 Kv2.1，和K+通道一般一样，单独作为K+传导的内在电决定因素 动脉肌细胞的膜特性。在此模型中，Kv2.1通道具有增加的物理作用 集群，从而协作选通Cav1.2通道。我们的数据表明， Kv2.1通道微调膜电位、Cav1.2聚集、 这些通道的功能耦合，从而导致钙离子内流，肌源性张力，最终影响血压。一个 突显我们工作重要性的关键发现是Kv2.1的表达因性别而异，导致 雌雄动脉肌细胞的钙离子内流和肌张力有显著差异。这个 我们互补的技能组合使我们能够实施多尺度系统方法， 涉及使用细胞、分子、生物物理、成像、基因编辑和全动物方法来 严格调查Kv2.1和Cav1.2组织的控制机制，以及它们对细胞的影响， 器官和全身在生理条件下的功能。该项目有三个具体目标。目标1是 确定Kv2.1表达水平改变对Cav1.2通道聚集和活性的影响，以及 动脉平滑肌和血压上的肌源性张力。目标2是定义潜在的机制 Kv2.1介导的Cav1.2功能的调节。最后，目标3是使用新的遗传模型来定义细胞 Kv2.1在调节Cav1.2中的自主作用及其可分离的传导和非传导功能 功能，动脉平滑肌细胞的生肌反应，以及全身血压。这个 拟议的研究有可能改变我们对离子通道如何组织的理解 血管平滑肌，并提供对动脉直径和血压如何不同的见解 在女性和男性之间进行调节。

Project Summary Dihydropyridine-sensitive, L-type Cav1.2 and delayed rectifier Kv2.1 channels play critical roles in the regulation of excitability and contraction in arterial smooth muscle. A salient feature of Cav1.2 channels is that they form clusters within which they undergo dynamic, reciprocal interactions that allow functional coupling of adjacent channels and thus amplification of Ca2+ signaling, which is critical to the development of myogenic tone. At present, however, the mechanisms controlling Cav1.2 clustering are unknown. The Trimmer and Santana labs have joined forces to address this fundamental issue. New preliminary data from our labs suggest a novel model that represents a paradigm shift relative to the generally accepted canonical role of Kv2.1, and K+ channels in general, as acting solely as K+ conducting electrical determinants of the intrinsic membrane properties of arterial myocytes. In this model, the Kv2.1 channel has a physical role to increase clustering and thus cooperative gating of Cav1.2 channels. Our data indicate that the balance between the separable electrical and structural roles of Kv2.1 channels fine tunes membrane potential, Cav1.2 clustering, functional coupling of these channels, and hence Ca2+ influx, myogenic tone, and, ultimately, blood pressure. A key finding that underscores the significance of our work is that Kv2.1 expression varies with sex, leading to significant differences in Ca2+ influx and myogenic tone between female and male arterial myocytes. The combination of our complementary skill sets allows us to implement a multi-scale systems approach that involves the use of cellular, molecular, biophysical, imaging, gene editing and whole-animal approaches to rigorously investigate the mechanisms controlling Kv2.1 and Cav1.2 organization, and how they impact cell, organ, and whole-body functions under physiological conditions. The project has three specific aims. Aim 1 is to determine the impact of altered Kv2.1 expression levels on clustering and activity of Cav1.2 channels, and myogenic tone in arterial smooth muscle, and on blood pressure. Aim 2 is to define the mechanisms underlying Kv2.1-mediated regulation of Cav1.2 function. Finally, Aim 3 is to use novel genetic models to define the cell autonomous role of Kv2.1, and its separable conducting and non-conducting functions, in regulating Cav1.2 function, and the myogenic response in arterial smooth muscle cells, and systemic blood pressure. The proposed studies have the potential of transforming our understanding of how ion channels are organized in vascular smooth muscle, and provide insights into how arterial diameter and blood pressure are differentially regulated in females versus males.