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-树突状细胞CaV1.2 L型电压门控Ca 2+通道（LTCC）在兴奋-转录（E-T）偶联中发挥作用。打开LTCC的去极化刺激通过Ca 2+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转录调节是学习和记忆基础的长期形式的兴奋性突触可塑性所必需的，例如晚期长时程增强（L-LTP）。因此，了解如何控制LTCC活动和信号传导以促进有效、特异性的突触与核通讯至关重要。突触到细胞核信号传递的主要问题是树突中产生的局部Ca 2+信号如何远程传递到索马中的细胞核。在上一个资助期间，我们建立了突触后支架蛋白A激酶锚定蛋白（AKAP）79/150，它通过修饰的亮氨酸拉链（LZ）基序与CaV 1.2结合，并锚定cAMP依赖性蛋白激酶（PKA）和钙调蛋白（Ca 2 +-CaM）激活的蛋白磷酸酶-2B（钙调磷酸酶; 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LTCC电流增强，这与激活AKAP锚定的CaN的Ca 2+负反馈回路强烈反对，有利于快速的钙依赖性失活（CDI）。此外，我们发现，本地LTCC激活AKAP锚定的CaN所需的NFAT易位到细胞核和转录响应去极化。然而，关键的问题仍然是关于AKAP 79/150是否调节LTCC的Ca 2+内流，特别是在树突棘响应谷氨酸受体激活和突触后的Ca 2+信号是否限制在树突可以局部激活CaN-NFAT信号到核。我们将探索这些问题，使用全细胞LTCC电流记录，局部谷氨酸释放，Ca 2+成像（Aim 1），NFAT成像（Aim 2），转录分析和L-LTP的细胞外记录（Aim 3）的组合。在所有三个目标中，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）AKAP 79突变体或使用来自AKAP 150 delta-PIX和delta-PKA敲入小鼠的神经元来研究AKAP 79/150对LTCC活性和NFAT信号传导的调节。总的来说，这个项目将测试一个中心的假设，在突触到核的通信，突触后钙信号在树突局部解码，然后有效地传递到核控制基因表达与突触可塑性。

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.