Modeling Beta2 receptor activity in cellular environment

描述(由申请人提供)： 环磷酸腺苷(CAMP)水平的变化将信息传递到下游效应分子，包括蛋白激酶A(PKA)和环核苷酸门控(CNG)通道。反过来，这些酶调节不同的细胞反应，如钙内流、兴奋性和基因表达。众所周知，cAMP信号的定位和频率有助于协调多种细胞功能，但对这些信号的亚细胞定位或动力学知之甚少。这个项目的总体目标是阐明cAMP信号定位的分子和细胞机制，cAMP信号的频率内容，以及cAMP振荡在细胞功能中的潜在作用。解决这些问题需要一种创新的方法来测量单细胞和细胞内的cAMP水平。为此，我们开发了基于基因工程CNG通道的高分辨率cAMP传感器。这些传感器以前所未有的空间和时间分辨率测量表面膜附近的cAMP信号。以下具体目标概述了应用该方法研究新生儿心肌细胞中cAMP信号的亚细胞定位和频率含量的计划。目的1.确定哪些PDE类型调节不同药物触发的cAMP信号，以及抑制不同类型的PDE如何影响cAMP信号的动力学。目的2.确定扩散屏障、PDE活性和PKA缓冲在定位cAMP信号中的相对贡献。目的3.建立描述cAMP信号在细胞内的空间扩散和动力学的数学模型。目的4.建立细胞环境中β2受体激活和脱敏的综合数学模型。建议的研究尤其与心肌细胞相关。β-肾上腺素能信号、cAMP产生、心脏兴奋性和疾病之间的密切关系已有很好的文献记载。然而，围绕β1和β2肾上腺素能受体的作用、“切换”、Gs和Gi的不同激活以及反应的区隔，存在着很大的争议。检测由激活特定GPCRs(例如，β2-肾上腺素能受体)或抑制磷酸二酯酶活性的试剂所触发的单细胞cAMP信号将为了解这些酶的生理功能及其与心脏功能的关系提供新的线索。重要的是，准确描述β2-肾上腺素能受体脱敏的综合数学模型的发展将使我们更好地了解药物如β-受体阻滞剂、反向激动剂和哮喘药物对信号网络和细胞生理学的影响。

DESCRIPTION (provided by applicant): Changes in cyclic AMP (cAMP) levels transmit information to downstream effectors including protein kinase A (PKA) and cyclic nucleotide-gated (CNG) channels. In turn, these enzymes regulate such diverse cellular responses as Ca2+ influx, excitability, and gene expression. It is accepted that the localization and frequency content of cAMP signals help to orchestrate a wide variety of cellular functions, yet little is known about either the sub-cellular localization or dynamics of these signals. The overall goal of this project is to elucidate the molecular and cellular mechanisms that localize cAMP signals, the frequency content of cAMP signals, and the potential roles of cAMP oscillations in cellular function. Addressing these issues will require an innovative approach for measuring cAMP levels in single cells and. To this end, we have developed high-resolution cAMP sensors based on genetically-engineered CNG channels. These sensors measure cAMP signals near the surface membrane with unprecedented spatial and temporal resolution. The following Specific Aims outline a plan to apply this approach to study the sub-cellular localization and frequency content of cAMP signals in neonatal cardiac myocytes. Aim 1. Determine which PDE types regulate cAMP signals triggered by different agents and how inhibition of different PDE types affects the kinetics of cAMP signals. Aim 2. Determine the relative contributions of diffusional barriers, PDE activity, and buffering by PKA in localizing cAMP signals. Aim 3. Develop mathematical models describing the spatial spread and kinetics of cAMP signals throughout the cell. Aim 4. Develop integrated mathematical models of the activation and desensitization of beta2ARs in the cellular environment. The proposed studies are particularly relevant in cardiac myocytes. The intimate relationships between beta-adrenergic signaling, cAMP production, cardiac excitability, and disease are well documented. However, there is a great deal of controversy surrounding the roles of beta1- and beta2-adrenergic receptors, 'switching', differential activation of Gs and Gi, and compartmentation of responses. Measuring single-cell, cAMP signals triggered by agents that activate specific GPCRs (e.g., beta2-adrenergic receptors) or inhibit phosphodiesterase activity will shed new light on the physiologic functions of these enzymes and their relation to cardiac function. Importantly, the development of integrated mathematical models that accurately describe beta2-adrenergic receptor desensitization will give us a better understanding of the impact of pharmacological agents such as beta-blockers, inverse agonists, and asthma drugs on signaling networks and cellular physiology.