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

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认知障碍在精神上的作用 迟缓、衰老与阿尔茨海默病S：L型电压门控引起的钙离子内流 钙通道(LTCC)可以影响突触可塑性和神经元的长期变化 通过打开和关闭细胞核中的基因转录来生存。虽然众所周知，信令 LTCC活性和基因的调节都需要非常接近的钙离子内流部位 表达，组织通道近端信号并转导它们的分子机制 在很大程度上是未知的。LTCC在神经元中活动的一个重要途径 调节涉及b-肾上腺素能受体介导的cAMP产生的刺激 腺苷环化酶与蛋白激酶A的激活。先前对心脏的研究表明 PKA对LTCC活性的有效调节需要通道蛋白和 通过与A-激酶锚定蛋白(AKAP)结合在通道附近定位PKA。 然而，人们对AKAP的作用或蛋白质的相反作用知之甚少 神经细胞LTCC调节中的磷酸酶。在突触后神经元中，一种可能发挥作用的AKAP 在调节LTCC磷酸化和核内转录因子信号转导中的关键作用 是AKAP79/150。我们的总体假设是，AKAP79/150针对PKA和CAN到LTCC，以 双向调节通道活动和向细胞核发出的信号。我们将对此进行测试 锚定的CAN强烈反对cAMP-PKA模型背景下的假设 对通道电流的调节起到钙离子负反馈机制的作用。在……里面 此外，我们将探索PKA和CAN在AKAP79/150中动态锚定的新角色 这些质膜定位于也控制下游激活的钙信号事件 NFAT和CREB转录因子。因此，我们的研究将表征一种新的分子 协调质膜LTCC钙信号以调节局部和 在神经元可塑性中起重要作用的远端反应。我们将使用生化、细胞生物学 和电生理方法在HEK-293细胞和海马神经元中的研究 AKAP79/150-LTCC调控：(目标1)DIRECT的分子和功能特征 AKAP79/150与LTCC CaV1.2在神经元通道调节中的相互作用 动态PKA和CaN锚定到AKAP79/150在神经元LTCC调节中的作用(目标3) AKAP79/150通道相关信号复合体在调节神经元LTCC中的作用 激发-转录耦合。

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.