Ca2+ Sparks as Regulators of Airway Contractility

描述（由申请人提供）：本项目旨在了解气道平滑肌（ASM）收缩性的控制和调节机制。目前的建议侧重于高度局部化和短暂的Ca2+事件（Ca2+火花），由肌浆网膜上的ryanodine受体（RyRs）打开引起，以及它们与质膜上附近的Ca2+激活离子通道的相互作用。与血管平滑肌一样，Ca2+火花激活火花微域中少量的大电导K+ (BK)通道，产生自发瞬态向外电流（STOCs），使ASM中的膜超极化。最近，我们已经确定了Ca2+火花在ASM中的另一个重要靶点，即Ca2+激活的CI- (CIca)通道，它引起自发瞬态内向电流（tics），从而使膜去极化。因此，本提案的中心假设是Ca2+火花协调膜通道的激活来调节气道收缩。利用高速数字Ca2+成像和同时贴片夹紧，以及2D和3D蛋白定位的综合方法，将应用于正常和转基因小鼠模型。此外，一种生理气道准备，即肺切片，将被用来研究Ca2+火花在调节气道本身收缩性中的作用。我们的具体目标是利用我们新开发的信号质量方法揭示Ca2+火花下RyRs的生物物理学（Aim 1）；确定RyRs与BK通道和CIca通道的功能和空间关系（目标2和3）；并确定Ca2+火花在气道中的生理作用（Aim 4）。我们期望这些研究将提供新的知识，从而导致哮喘和其他支气管痉挛疾病的新治疗方法的发展。

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.