AKAP Regulation of Neuronal L-type Calcium Channel Signaling to the Nucleus

描述（由申请人提供）：在海马神经元中，体细胞-树突CaV1.2 l型电压门控Ca2+通道（LTCC）在兴奋-转录（E-T）耦合中起作用。打开ltcc的去极化刺激通过Ca2+调节的激酶和磷酸酶激活转录因子camp反应元件结合蛋白（CREB）和活化t细胞核因子（NFAT）。重要的是，LTCC的转录调节是学习和记忆基础的兴奋性突触可塑性的持久形式所必需的，如后期长期增强（L-LTP）。因此，了解LTCC活性和信号是如何被控制以促进有效的、特定的突触到细胞核的通信是至关重要的。突触到细胞核信号传导的主要问题是树突中产生的局部Ca2+信号如何远程传递到体细胞中的细胞核。在上一个资助期内，我们建立了突触后支架蛋白a激酶锚定蛋白（AKAP） 79/150，该蛋白通过修饰的亮氨酸拉链（LZ）基元与CaV1.2结合，锚定camp依赖性蛋白激酶（PKA）和Ca2+-钙调蛋白（CaM）激活的蛋白磷酸酶2b (calcineurin; CaN)，作为神经元LTCC电流和NFAT激活的重要调节剂。我们发现，akap锚定的PKA促进了LTCC电流的增强，这与激活akap锚定的CaN的Ca2+负反馈环强烈反对，有利于快速的钙依赖性失活（CDI）。此外，我们发现，在去极化反应中，NFAT易位到细胞核和转录需要akap锚定的CaN的局部LTCC激活。然而，关于AKAP79/150是否在响应谷氨酸受体激活时特异性调节树突棘LTCC Ca2+内流以及树突中限制的突触后Ca2+信号是否可以局部激活can - nfat信号传导到细胞核的关键问题仍然存在。我们将结合全细胞LTCC电流记录、局部谷氨酸释放、Ca2+成像（Aim 1）、NFAT成像（Aim 2）、转录分析和L-LTP的细胞外记录（Aim 3）来探讨这些问题。在这三个目标中，将通过在大鼠神经元中表达PKA锚定缺陷（delta-PKA）、CaN锚定缺陷（delta-PIX）和LZ结构域（delta-LZ） AKAP79突变体或使用AKAP150 δ - pix和delta-PKA敲入小鼠的神经元来研究AKAP79/150对LTCC活性和NFAT信号传导的调节。总的来说，该项目将测试突触到细胞核通信的一个中心假设，即突触后Ca2+信号在树突中局部解码，然后有效地传递到细胞核，以控制与突触可塑性相关的基因表达。

DESCRIPTION (provided by applicant): In hippocampal neurons, somato-dendritic CaV1.2 L-type voltage-gated Ca2+ channels (LTCC) function in excitation-transcription (E-T) coupling. Depolarizing stimuli that open LTCCs activate the transcription factors cAMP-response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT) through Ca2+-regulated kinase and phosphatases. Importantly, LTCC transcriptional regulation is required for long-lasting forms of excitatory synaptic plasticity that underlie learning and memory, such as late-phase long-term potentiation (L-LTP). Thus, it is crucial to understand how LTCC activity and signaling are controlled to promote efficient, specific synapse to nucleus communication. The primary question in synapse-to-nucleus signaling is how local Ca2+ signals generated in dendrites are relayed remotely to the nucleus in the soma. In the last funding period, we established the postsynaptic scaffold protein A-kinase anchoring protein (AKAP) 79/150, which binds to CaV1.2 through a modified leucine zipper (LZ) motif and anchors the cAMP-dependent protein kinase (PKA) and Ca2+-calmodulin (CaM)-activated protein phosphatase-2B (calcineurin; CaN), as an essential regulator of neuronal LTCC currents and NFAT activation. We found that AKAP-anchored PKA promoted LTCC current enhancement that was strongly opposed by a Ca2+ negative feedback loop activating AKAP-anchored CaN to favor rapid, calcium-dependent inactivation (CDI). In addition, we found that local LTCC activation of AKAP-anchored CaN was required for NFAT translocation to the nucleus and transcription in response to depolarization. However, key questions remain regarding whether AKAP79/150 regulates LTCC Ca2+ influx specifically in dendritic spines in response to glutamate receptor activation and whether postsynaptic Ca2+ signals restricted in dendrites can locally activate CaN-NFAT signaling to the nucleus. We will explore these questions using a combination of whole-cell LTCC current recordings, local glutamate uncaging, Ca2+ imaging (Aim 1), NFAT imaging (Aim 2), transcriptional analyses, and extracellular recordings of L-LTP (Aim 3). In all three aims, AKAP79/150 regulation of LTCC activity and NFAT signaling will be investigated by expressing PKA anchoring deficient (delta-PKA), CaN anchoring deficient (delta-PIX), and LZ domain (delta-LZ) AKAP79 mutants in in rat neurons or using neurons from AKAP150 delta-PIX and delta-PKA knock-in mice. Overall, this project will test a central hypothesis in synapse-to-nucleus communication that postsynaptic Ca2+ signals are locally decoded in dendrites and then efficiently relayed to the nucleus to control gene expression linked to synaptic plasticity.