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

描述(申请人提供)：在海马神经元中，体树突状CaV1.2 L型电压门控性钙通道在兴奋-转录(E-T)偶联中起作用。开放LTCC的去极化刺激通过钙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细胞的核因子(NFAT)。重要的是，LTCC转录调控对于长期存在的兴奋性突触可塑性是必需的，这种可塑性是学习和记忆的基础，例如晚期长时程增强(L-LTP)。因此，了解LTCC的活动和信号如何被控制以促进高效、特异的突触到核的通讯是至关重要的。突触到核信号传递的主要问题是，在树突中产生的局部钙信号是如何远程传递到胞体内的核的。在上一个资助阶段，我们建立了突触后支架蛋白A-激酶锚定蛋白(AKAP)79/150，它通过一个改进的亮氨酸拉链(LZ)基序与CaV1.2结合，并锚定cAMP依赖的蛋白激酶(PKA)和钙调素(CaM)激活的蛋白磷酸酶2B(CaN；CaN)，作为神经元LTCC电流和NFAT激活的重要调节因子。我们发现，AKAP锚定的PKA促进了LTCC电流的增强，而激活AKAP锚定的CaN的钙离子负反馈环强烈反对这种增强，从而有利于快速的钙依赖失活(CDI)。此外，我们还发现，AKAP锚定的CaN的局部LTCC激活是NFAT移位到细胞核和转录对去极化的反应所必需的。然而，关键的问题仍然是AKAP79/150是否响应谷氨酸受体的激活而调节树突棘中的LTCC钙离子内流，以及限制在树突中的突触后钙信号是否可以局部激活到细胞核的CaN-NFAT信号。我们将结合全细胞LTCC电流记录、局部谷氨酸去化、钙离子成像(目标1)、非荧光成像(目标2)、转录分析和L-LTP(目标3)的细胞外记录来探索这些问题。在所有这三个AIMS中，AKAP79/150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 Delta-PIX和Delta-PKA敲入小鼠的神经元来研究AKAP79/150对LTCC活性和NFAT信号的调节。总体而言，该项目将测试突触到核通讯的一个中心假说，即突触后钙信号在树突中被局部解码，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.