Cyclic AMP signaling in cardiac myocytes

描述（由申请人提供）：环AMP （cAMP）水平的变化将信息传递给下游效应器，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ca2 +内流、兴奋性和基因表达等多种细胞反应。人们普遍认为cAMP信号的定位和频率内容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态知之甚少。该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中的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。

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 Ca 2+ 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. 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. Simultaneously measure cAMP concentration and PKA-mediated regulation of L-type Ca 2+ channel activity. 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, differential activation of Gs and Gi, and compartmentation of responses. Measuring single-cell, cAMP signals triggered by agents that activate specific GPCRs (e.g., protenoid, beta1-,or beta2-adrenergic receptors) or inhibit phosphodiesterase (PDE) will shed new light on the physiologic functions of these enzymes and their relation to cardiac function.