Modeling Beta2 receptor activity in cellular environment

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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.