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：
10363116
负责人：
Arturo Andrade
金额：
$ 38.62万
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/10363116
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 Cholecystokinin Clinical Cognition Communication Coupled Coupling Development Dose Electrophysiology (science)Emotional Excitatory Synapse Exhibits Exons Genes Glutamates Goals Hippocampus (Brain)Impaired cognition Impairment Inhibitory Synapse Interneurons Knowledge Label Learning Memory Molecular Morphine Neuraxis Neurons Pharmacology Physiology Probability Proteins RNA Splicing Receptor Inhibition Receptor Signaling Regulation Role S-nitro-N-acetylpenicillamine SNAP receptor Site Slice Synapses Testing Therapeutic Variant Work base biophysical properties dimer drug sensitivity gamma-Aminobutyric Acid hippocampal pyramidal neuron improved innovation insight mouse model mu opioid receptors 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的并且具有剂量依赖性。例如，低剂量会产生抗焦虑作用而高剂量会引起焦虑；对记忆和认知也有类似的影响。然而，其原因尚不清楚。在突触水平，大麻素受体 1 (CB1R) 已知会影响通过抑制突触前钙通道（包括 N 型 (CaV2.2) 通道）释放递质它们对于将神经元活动与海马体 (HPC) 中的递质释放耦合起来至关重要。这脑子该区域对于情绪处理和学习很重要，而 CaV2.2 通道对于大脑中研究最充分的回路之一至关重要。我们的长期目标是破译CaV2.2通道的调控和功能特定的 HPC 突触，以告知 HPC 活动的基本机制，并实现基于 CB1R 的新疗法发信号。选择性剪接是一种细胞特异性机制，会影响 CaV2.2 通道的功能和调节。外显子 18a 的剪接产生 +18a-CaV2.2 和 D18a-CaV2.2 剪接变体。神经传递在表达 +18a-CaV2.2 变体的突触表现出递质释放的可能性增强，并降低与表达 +18a-CaV2.2 变体的激动剂相比，CB1R 激动剂的调节有所不同，但潜在的机制尚不清楚。 +18-CaV2.2 剪接变体在该区域包含 21 个氨基酸插入与释放机械相互作用。我们的中心假设是 +18a-CaV2.2 剪接变体增强递质通过与 SNARE 蛋白的差异相互作用释放并阻止 CB1R 对神经传递的抑制。到对此进行测试，我们将使用经过验证的小鼠模型，其剪接选择受到限制（仅 +18a 或仅 18a），并且基于重组酶的 HPC 中特定神经元标记，用于急性切片的电生理学和生化评估蛋白质相互作用的测定。该项目的具体目标是： 1) 确定功能影响 +18a-CaV2.2 和 D18a-CaV2.2 剪接变体对 HPC 中发射器释放的影响，以及 2) 确定 HPC 中发射器释放的 CB1R 调制上的 +18a-CaV2.2 和 D18a-CaV2.2 拼接变体。结果该项目预计将提供对海马功能基本机制的见解大麻素的矛盾作用。 CB1R 信号传导的新型细胞特异性效应器可以积极地影响基于大麻素的疗法的发展