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

• 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
• 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

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，并依赖于剂量。例如，低剂量产生抗焦虑作用 而高剂量则会引起焦虑;对记忆和认知也有类似的影响。但 其原因尚不清楚。在突触水平，已知大麻素受体1（CB 1 R）影响 通过抑制突触前钙通道（包括N型（CaV2.2）通道）释放递质 在将神经元活动与海马体（HPC）中的递质释放耦合中至关重要。这个大脑 该区域对于情绪处理和学习非常重要，CaV2.2通道是大脑中研究最多的回路之一。我们的长期目标是破译CaV2.2通道的调节和功能， 特异性HPC突触，以告知HPC活性的基本机制，并实现基于CB 1 R的新型疗法 发信号。 选择性剪接是影响CaV2.2通道功能和调节的细胞特异性机制。 外显子18 a的剪接产生+18 a-CaV 2.2和D18 a-CaV 2.2剪接变体。神经传递 表达+18 a-CaV 2.2变体的突触表现出增强的递质释放概率，和降低的 与表达+18 a-CaV 2.2变异体的那些相比，CB 1 R激动剂的调节，但潜在的 机制不明。+18-CaV2.2剪接变体在该区域中含有21个氨基酸插入， 与释放机构相互作用。我们的中心假设是+18 a-CaV 2.2剪接变异体增强了信号传导， 通过与SNARE蛋白的差异相互作用释放和阻止CB 1 R对神经传递的抑制。到 为了验证这一点，我们将使用经过验证的小鼠模型，其具有有限的剪接选择（仅+18 a或仅18 a）， 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物化学 分析以评估蛋白质相互作用。该项目的具体目标是：1）确定功能影响 + 18 a-CaV 2.2和D18 a-CaV 2.2剪接变异体对HPC中递质释放的作用，以及2）确定 + 18 a-CaV 2.2和D18 a-CaV 2.2剪接变体对HPC中CB 1 R调节递质释放的影响。的结果 该项目也有望为海马功能的基本机制提供深入的见解， 大麻素的矛盾作用CB 1 R信号传导的新型细胞特异性效应物可以积极地 基于大麻素治疗剂的影响发展