CaV2.2 splice variants in the hippocampus: function and pharmacology

海马 CaV2.2 剪接变异体：功能和药理学

基本信息

批准号：
10652276
负责人：
Arturo Andrade
金额：
$ 38.25万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10652276
关键词：
Absence of pain sensation Action Potentials Acute Agonist Alternative Splicing Amino Acids Anxiety Anxiety Disorders Area Axon Biochemical Biochemistry Biological Assay Brain CNR1 gene Calcium Calcium Channel Cannabinoids Cells Central Nervous System Cholecystokinin Clinical Cognition Communication Coupled Coupling Development Dose Electrophysiology (science)Emotional Excitatory Synapse Exhibits Exons Genes Glutamates Goals Hippocampus Impaired cognition Impairment Inhibitory Synapse Interneurons Knowledge Label Learning Memory Molecular Morphine Neurons Opioid Receptor Pharmacology Physiology Probability Proteins RNA Splicing Recreation Regulation Role SNAP receptor Shapes Signal Transduction Site Slice Synapses Testing Therapeutic Variant Work biophysical properties dimer drug sensitivity gamma-Aminobutyric Acid hippocampal pyramidal neuron improved innovation insight mouse model neurotransmission new therapeutic target novel novel therapeutics presynaptic prevent recombinase recruit response selective expression syntaxin 1A

项目摘要

The effects of cannabinoids are diverse and dose dependent. For instance, low doses produce anxiolysis whereas high doses induce anxiety; similar effects have been seen on memory and cognition. However, the reasons for this are not clear. At the synapse level, the cannabinoid receptor 1 (CB1R) is known to impact transmitter release through inhibition of presynaptic calcium channels, including the N-type (CaV2.2) channels that are paramount in coupling neuronal activity to transmitter release in the hippocampus (HPC). This brain area is important for emotional processing and learning, and CaV2.2 channels are essential in one of the best-studied circuits in the brain. Our long-term goal is to decipher the regulation and function of CaV2.2 channels at specific HPC synapses to inform basic mechanisms of HPC activity, and enable novel therapies based on CB1R signaling. Alternative splicing is a cell-specific mechanism that impacts the function and regulation of CaV2.2 channels. Splicing of exon 18a generates the +18a-CaV2.2 and D18a-CaV2.2 splice variants. Neurotransmission in synapses that express +18a-CaV2.2 variants exhibit enhanced probability of transmitter release, and reduced modulation by CB1R agonists compared to those that express the +18a-CaV2.2 variants, but the underlying mechanisms are unknown. +18-CaV2.2 splice variants contain a 21 aminoacid insertion in the region that interacts with the release machinery. Our central hypothesis is +18a-CaV2.2 splice variants enhance transmitter release and prevent CB1R inhibition of neurotransmission via differential interaction with SNARE proteins. To test this, we will use validated mouse models with restricted splice choice (+18a-only or 18a-only) and recombinase-based labeling of specific neurons in HPC for electrophysiology in acute slices, and biochemical assays to evaluate protein interaction. The specific aims of the project are: 1) To determine the functional impact of +18a-CaV2.2 and D18a-CaV2.2 splice variants on transmitter release in HPC, and 2) To determine the role of +18a-CaV2.2 and D18a-CaV2.2 splice variants on CB1R modulation of transmitter release in HPC. The results of this project are expected to provide insights into the basic mechanisms underlying hippocampal function, as well for the contradictory effects of cannabinoids. Novel cell-specific effectors of CB1R signaling could positively impact development of cannabinoid-based therapeutics

大麻素的作用是多种多样和剂量依赖性的。例如，低剂量产生焦虑分析而高剂量会引起焦虑；对记忆和认知也有类似的影响。但是，原因尚不清楚。在突触水平上，已知大麻素受体1（CB1R）会影响通过抑制突触前钙通道的释放，包括N型（CAV2.2）通道这对于在海马（HPC）中释放神经元活性的耦合至关重要。这个大脑区域对于情绪处理和学习很重要，而Cav2.2渠道在大脑中研究最佳的电路之一中至关重要。我们的长期目标是破译Cav2.2通道的调节和功能特定的HPC突触为HPC活性的基本机制提供信息，并基于CB1R启用新型疗法信号。替代剪接是一种影响CAV2.2通道功能和调节的细胞特异性机制。外显子18a的剪接生成 +18A-CAV2.2和D18A-CAV2.2剪接变体。神经传递表达 +18a-cav2.2变体的突触表现出增强的发射机释放概率，并降低与表达 +18a-cav2.2变体的调节器相比，CB1R激动剂的调节，但基础机制是未知的。 +18-cav2.2剪接变体在该区域中包含21个氨基酸插入与释放机械进行交互。我们的中心假设是 +18A-CAV2.2剪接变体增强发射器释放并防止CB1R抑制神经传递，通过与SNARE蛋白的差异相互作用。到对此进行测试，我们将使用具有限制性剪接选择的经过验证的鼠标模型（仅+18A或仅18A），并且 HPC中特定神经元的基于重组酶的标记，用于急性切片中的电生理学和生化评估蛋白质相互作用的测定。该项目的具体目的是：1）确定功能影响在HPC中释放发射机上的 +18a-Cav2.2和D18A-CAV2.2剪接变体，2）确定 +18A-CAV2.2和D18A-CAV2.2剪接变体在HPC中发射器释放的CB1R调制。结果预计该项目将提供有关海马功能基本机制的见解对于大麻素的矛盾作用。 CB1R信号的新细胞特异性效应子可以积极地影响基于大麻素治疗剂的开发