Ca2+ Sparks as Regulators of Airway Contractility

描述(由申请者提供)：该项目旨在了解控制和调节呼吸道平滑肌(ASM)收缩的机制。目前的研究集中在肌浆网膜兰尼定受体(RyRs)开放引起的高度局部化和短暂的钙离子事件(钙火花)，以及它们与质膜上附近的钙激活离子通道的相互作用。与血管平滑肌一样，钙火花激活火花微区的少量大电导K(BK)通道，产生自发的瞬时外向电流(STOCs)，使ASM膜超极化。最近，我们在ASM中发现了钙电火花的另一个重要靶点，即钙激活的CI-(CICA)通道，它能引起自发的瞬时内向电流(STICs)，从而使膜去极化。因此，这一建议的中心假设是，钙火花协调膜通道的激活，以调节呼吸道收缩能力。一种使用高速数字钙成像和同时膜片钳以及2D和3D蛋白质定位的综合方法将应用于正常和转基因小鼠模型。此外，生理性的呼吸道准备，即肺切片，将被用来研究钙火花在调节呼吸道本身收缩性能方面的作用。我们的具体目标是用我们新开发的信号质量方法(目标1)揭示钙火花背后的RyRs的生物物理学；确定RyR与BK通道和CICA通道的功能和空间关系(目标2和3)；以及确定钙火花在呼吸道中的生理作用(目标4)。我们希望这些研究将提供新的知识，可能导致开发新的治疗方法来治疗哮喘和其他支气管炎疾病。

DESCRIPTION (provided by applicant): This project seeks to understand the mechanisms by which the contractility of airway smooth muscle (ASM) is controlled and regulated. The present proposal focuses on the highly localized and short-lived Ca2+ events (Ca2+ sparks), resulting from the opening of ryanodine receptors (RyRs) in the sarcoplasmic reticulum membrane, and their interactions with nearby Ca2+-activated ion channels in the plasma membrane. As in vascular smooth muscle, Ca2+ sparks activate a small number of large-conductance K+ (BK) channels in the spark microdomain to generate spontaneous transient outward currents (STOCs) which hyperpolarize the membrane in ASM. Recently we have identified another important target of Ca2+ sparks in ASM, i.e. Ca2+-activated CI- (CIca) channels which cause spontaneous transient inward currents (STICs) and thus depolarize the membrane. Accordingly, the central hypothesis of this proposal is that Ca2+ sparks coordinate the activation of membrane channels to regulate airway contractility. An integrated approach using high-speed digital Ca2+ imaging with simultaneous patch-clamping, and also 2D and 3D protein localization, will be applied using normal and transgenic mouse models. Furthermore, a physiological airway preparation, i.e., lung slices, will be employed to investigate the role of Ca2+ sparks in regulating contractility of airways themselves. Our specific objectives are to uncover the biophysics of RyRs underlying Ca2+ sparks using our newly developed signal mass approach (Aim 1); to determine the functional and spatial relationships of RyRs to BK channels and CIca channels (Aims 2 and 3); and to determine the physiological role of Ca2+ sparks in airways (Aim 4). We expect that these studies will provide new knowledge which could lead to development of novel therapeutic approaches for asthma and other bronchospasitc disorders.