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

描述(申请人提供)：阐明神经元存活和可塑性的调控机制对于了解正常的学习和记忆以及智力低下、衰老和阿尔茨海默氏症的认知障碍至关重要。钙离子通过L类型的电压门控钙通道(LTCC)内流可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LTCC活性和基因表达都需要非常靠近钙内流部位的信号来调节，但组织通道近端信号并将其传导到细胞核的分子机制在很大程度上尚不清楚。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酰环化酶刺激cAMP的产生和激活PKA。以往的心脏研究表明，PKA对LTCC活性的有效调节需要通道蛋白的磷酸化，并通过与A-激酶锚定蛋白(AKAP)的结合将PKA定位在通道附近。然而，对AKAP的作用或蛋白磷酸酶在神经元LTCC调节中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磷酸化和向核内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靶向PKA和CaN到LTCC，以双向调节通道活动和到细胞核的信号。我们将在一个模型的背景下检验这一假设，在该模型中，锚定可以强烈反对cAMP-PKA对通道电流的调节，以发挥钙离子负反馈机制的作用。此外，我们还将探索在这些质膜定位的钙信号事件中，PKA和CaN动态锚定到AKAP79/150上的新角色，这些信号事件也控制着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钙信号来调节在神经元可塑性中重要的局部和远端反应。我们将利用生化、细胞生物学和电生理学的方法，在HEK-293细胞和海马神经元中研究AKAP79/150-LTCC的调控：(1)AKAP79/150与LTCC CaV1.2之间直接相互作用在神经元通道调节中的分子和功能表征；(2)动态PKA和CaN锚定到AKAP79/150在神经元LTCC调节中的作用；(3)AKAP79/150通道相关信号复合体在调节神经元LTCC激发-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.