Does alternative splicing regulate G protein inhibition of calcium channels?

选择性剪接是否调节 G 蛋白对钙通道的抑制？

• 批准号: 7808529
• 负责人: Cecilia Phillips Toro
• 金额: $ 2.85万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7808529
• 关键词: Affect; Agonist; Alternative Splicing; Binding; Calcium; Calcium Channel; Calcium Channel Inhibition; Cells; Communication; Coupling; Dopamine D1 Receptor; Electrophysiology (science); Engineering; Exocytosis; Exons; G alpha q Protein; G-Protein-Coupled Receptors; GTP-Binding Proteins; Mediating; Midbrain structure; Molecular; Mus; N-Type Calcium Channels; Nerve Degeneration; Neurons; Neurotransmitters; Parkinson Disease; Peptides; Peripheral; Pharmacology; Predisposition; Presynaptic Terminals; Protein Inhibition; Proteins; RNA Splicing; Receptor, Angiotensin, Type 1; Regulation; Research Project Grants; Role; Small Interfering RNA; Specificity; System; Variant; Work; dopaminergic neuron; mRNA Precursor; neurotransmission; novel; presynaptic; voltage

DESCRIPTION (provided by applicant): N-type (CaV2.2) voltage-gated calcium channels (VGCCs) are expressed at presynaptic terminals in virtually all central and peripheral neurons where they control the calcium entry that triggers exocytosis. Therefore, the efficacy of neurotransmission is directly correlated with the amount of calcium that passes through VGCCs. This relationship is exploited by a classic form of presynaptic regulation: G protein-mediated inhibition of N-type channels. The molecular rules governing the specific coupling of G protein-coupled receptors (GPCRs) to calcium channels are not fully understood. Recent work from the Lipscombe lab suggests an exciting and novel explanation for channel-G protein interaction specificity: alternative splicing of CaV2.2 pre-mRNA is the molecular mechanism that controls G protein coupling to the N-type calcium channel. This proposal focuses on the role of an alternative exon, el 8a, in the G protein-mediated inhibition of the N-type channel. The overarching hypothesis of this proposal is: cell- specific inclusion of el 8a renders N-type channels susceptible voltage-independent inhibition by GPCRs. The specific aims of this project are to: (1) determine which G proteins couple to e18a; (2) determine how el 8a contributes to the inhibition of the N-type channel by the D1 dopamine receptor (D1R) and the Angiotensin II receptor type 1 (AT1R); (3) determine the role of el8a in mediating G protein-dependent inhibition of native N-type currents in dopaminergic neurons. N-type channel el 8a splice variants will be studied both in an expression system (tsA201 cells) and in dissociated neurons from mouse midbrain cultures engineered to express GFP in DIR-expressing neurons. The function of the channels will be probed using electrophysiology and pharmacology. G protein inhibition of cloned and native channels will be assessed with GTPgS or GPCR agonists. This inhibition will be disrupted by (1) specifically inhibiting N-type channels containing e18a with siRNAs, and (2) sequestering el 8a binding partners with an inhibitory peptide. Overall, this project serves to elucidate the role of alternative splicing of the N-type channel in controlling susceptibility to G protein-mediated inhibition. RELEVANCE: Neurodegeneration in Parkinson's Disease specifically affects midbrain neurons. Neurotransmitters can modulate the activity of these neurons by inhibiting calcium channels, proteins vital to communication between neurons. This research project serves to understand the inhibition of calcium channels specifically expressed in midbrain neurons.

描述（由申请人提供）：N型（CAV2.2）电压门控钙通道（VGCC）在几乎所有中心和周围神经元中的突触前终端表达，它们控制着触发胞吞作用的钙进入。因此，神经传递的功效与通过VGCC的钙量直接相关。这种关系是通过突触前调节的经典形式来利用的：G蛋白介导的N型通道的抑制作用。尚未完全了解管理G蛋白偶联受体（GPCR）与钙通道的特定偶联的分子规则。 Lipscombe实验室的最新工作提出了对通道G蛋白相互作用特异性的令人兴奋且新颖的解释：CAV2.2的替代剪接Pre-MRNA是控制G蛋白偶联到N型钙通道的分子机制。该提案的重点是替代外显子El 8a在G蛋白介导的N型通道抑制中的作用。该提议的总体假设是：EL 8A的细胞特异性包含使N型通道易受到GPCR的敏感电压抑制。该项目的具体目的是：（1）确定哪些G蛋白与E18A； （2）确定EL 8a如何通过D1多巴胺受体（D1R）和血管紧张素II受体1型（AT1R）抑制N型通道的抑制作用； （3）确定EL8A在介导G蛋白依赖性抑制天然N型电流中多巴胺能神经元中的作用。 N型通道EL 8A剪接变体将在表达系统（TSA201细胞）和来自小鼠中脑培养物中的分离神经元中进行研究，该神经元被设计为表达表达DIR表达神经元的GFP的小鼠中脑培养物。通道的功能将使用电生理学和药理学进行探测。 GTPGS或GPCR激动剂将评估G蛋白抑制的克隆和天然通道。 （1）特异性抑制含有E18A的N型通道和（2）用抑制性肽隔离EL 8A结合伴侣的n型通道会破坏这种抑制作用。总体而言，该项目旨在阐明N型通道在控制G蛋白介导的抑制易感性方面的替代剪接的作用。相关性：帕金森氏病的神经变性专门影响中脑神经元。神经递质可以通过抑制钙通道，对神经元之间的通信至关重要的蛋白质来调节这些神经元的活性。该研究项目旨在了解对中脑神经元特别表达的钙通道的抑制作用。