AKAP Anchored PKA and Calcineurin Regulation of Neuronal L-type Calcium Channels

描述（申请人提供）：阐明调节神经元存活和可塑性的机制与理解正常的学习和记忆以及智力迟钝，衰老和阿尔茨海默氏症的认知障碍有关。钙离子（Ca2+）通过l型电压门控钙通道（ltcc）的内流可以通过开启和关闭细胞核中的基因转录来影响突触可塑性和神经元存活的长期变化。虽然已知Ca2+内流位点附近的信号是调节LTCC活性和基因表达所必需的，但组织通道近端信号并将其转导到细胞核的分子机制在很大程度上是未知的。调节神经元LTCC活性的一个重要途径涉及b-肾上腺素能受体介导的腺苷酸环化酶和激酶PKA激活对cAMP产生的刺激。先前在心脏中的研究表明，PKA对LTCC活性的有效调节需要通道蛋白磷酸化，并通过与a -激酶锚定蛋白（AKAP）结合将PKA定位在通道附近。然而，关于akap在神经元LTCC调控中的作用或蛋白磷酸酶的相反作用知之甚少。在突触后神经元中，AKAP79/150可能在调节LTCC磷酸化和向细胞核转录因子发出信号方面发挥关键作用。我们的总体假设是，AKAP79/150将PKA和CaN靶向ltcc，双向调节通道活性和向细胞核发送信号。我们将在锚定CaN强烈反对cAMP-PKA调节通道电流作为Ca2+负反馈机制的模型背景下检验这一假设。此外，我们将探索PKA和CaN在这些质膜定位Ca2+信号事件中动态锚定到AKAP79/150的新作用，这些事件也控制着NFAT和CREB转录因子的下游激活。因此，我们的研究将描述一种新的分子组装，它协调质膜LTCC Ca2+信号来调节局部和远端反应，这在神经元可塑性中很重要。我们将在HEK-293细胞和海马神经元中使用生化、细胞生物学和电生理学方法来研究AKAP79/150-LTCC调控：（目的1）AKAP79/150与LTCC CaV1.2在神经元通道调控中的直接相互作用的分子和功能表征；（目的2）动态PKA和CaN锚定在AKAP79/150上在神经元LTCC调控中的作用；（目的3）AKAP79/150通道相关信号复合物在调节神经元LTCC兴奋-转录耦合中的作用。

DESCRIPTION (provided by applicant): Elucidating mechanisms regulating neuronal survival and plasticity is relevant for understanding normal learning and memory as well as cognitive impairments in mental retardation, aging and Alzheimer's. Influx of calcium ions (Ca2+) through L-type voltage-gated calcium channels (LTCCs) can influence long-term changes in synaptic plasticity and neuronal survival by turning on and off gene transcription in the nucleus. While it is known that signaling very near the site of Ca2+ influx is required for regulation of both LTCC activity and gene expression, molecular mechanisms that organize channel proximal signals and transduce them to the nucleus are largely unknown. One important pathway by which LTCC activity in neurons is regulated involves b-adrenergic receptor-mediated stimulation of cAMP production by adenylyl cyclase and activation of the kinase PKA. Previous studies in the heart suggest that efficient regulation of LTCC activity by PKA requires phosphorylation of the channel protein and localization of PKA near the channel through binding to A-kinase-anchoring proteins (AKAP). However, little is known about the roles of AKAPs or the opposing actions of protein phosphatases in neuronal LTCC regulation. In postsynaptic neurons one AKAP that may play a key role in regulating LTCC phosphorylation and signaling to transcription factors in the nucleus is AKAP79/150. Our overall hypothesis is that AKAP79/150 targets PKA and CaN to LTCCs to bi-directionally regulate channel activity and signaling to the nucleus. We will test this hypothesis in the context of a model in which anchored CaN strongly opposes cAMP-PKA regulation of the channel currents to function as a Ca2+ negative feedback mechanism. In addition, we will explore a novel role for dynamic anchoring of PKA and CaN to AKAP79/150 in these plasma membrane localized Ca2+ signaling events that also control downstream activation of NFAT and CREB transcription factors. Thus, our studies will characterize a novel molecular assembly that coordinates plasma membrane LTCC Ca2+ signaling to regulate both local and distal responses that are important in neuronal plasticity. We will use biochemical, cell biological and electrophysiological approaches in HEK-293 cells and hippocampal neurons to study AKAP79/150-LTCC regulation: (Aim 1) Molecular and functional characterization of a direct interaction between AKAP79/150 and the LTCC CaV1.2 in neuronal channel regulation; (Aim 2) Role of dynamic PKA and CaN anchoring to AKAP79/150 in neuronal LTCC regulation; (Aim 3) Role of the AKAP79/150 channel-associated signaling complex in regulating neuronal LTCC excitation-transcription coupling.