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

说明（由申请人提供）：阐明调节神经元存活和可塑性的机制与理解正常学习和记忆以及智力迟钝、衰老和阿尔茨海默氏症中的认知障碍有关。钙离子（Ca 2+）通过L型电压门控钙通道（LTCCs）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。虽然它是已知的，信号非常接近的网站的Ca 2+内流的LTCC活性和基因表达的调节所需的，分子机制，组织通道近端信号，并将它们传递到细胞核是很大程度上未知的。调节神经元中LTCC活性的一个重要途径涉及β-肾上腺素能受体介导的腺苷酸环化酶对cAMP产生的刺激和激酶PKA的活化。心脏中的先前研究表明，PKA对LTCC活性的有效调节需要通道蛋白的磷酸化和PKA通过与A-激酶锚定蛋白（AKAP）结合而定位在通道附近。然而，很少有人知道AKAP的作用或蛋白磷酸酶在神经元LTCC调节的相反行动。在突触后神经元中，AKAP 79/150可能在调节LTCC磷酸化和向核中转录因子发出信号中起关键作用。我们的总体假设是AKAP 79/150靶向PKA和CaN到LTCC以双向调节通道活性和向细胞核的信号传导。我们将测试这一假设的背景下，锚定的钙离子强烈反对cAMP-PKA调节通道电流的钙离子负反馈机制的模型。此外，我们将探索PKA和CaN动态锚定AKAP 79/150在这些质膜定位的Ca 2+信号传导事件，也控制下游激活NFAT和CREB转录因子的新作用。因此，我们的研究将表征一种新的分子组装体，其协调质膜LTCC Ca 2+信号传导以调节在神经元可塑性中重要的局部和远端反应。我们将在HEK-293细胞和海马神经元中采用生物化学、细胞生物学和电生理学方法研究AKAP 79/150-LTCC调节：（目的1）在神经元通道调节中AKAP 79/150和LTCC CaV 1.2之间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AKAP 79/150在神经元LTCC调节中的作用;（目的3）AKAP 79/150通道相关信号复合物在神经元LTCC兴奋-转录偶联中的作用。

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.