Does alternative splicing regulate G protein inhibition of calcium channels?

描述(申请人提供)：N型(Cav2.2)电压门控钙通道(VGCC)在几乎所有中枢和外周神经元的突触前终末表达，在那里它们控制触发胞吐的钙内流。因此，神经传递的效果与通过VGCC的钙量直接相关。这种关系被突触前调节的一种经典形式所利用：G蛋白介导的对N型通道的抑制。控制G蛋白偶联受体(GPCRs)与钙通道特异性偶联的分子规律尚不完全清楚。Lipscombe实验室最近的工作对通道-G蛋白相互作用的特异性提出了一个令人兴奋的新解释：Cav2.2前-mRNA的选择性剪接是控制G蛋白与N型钙通道偶联的分子机制。本研究的重点在于另一个外显子el 8a在G蛋白介导的N型通道抑制中的作用。这一建议的主要假设是：细胞特异性地包含EL8a使N型通道容易受到GPCRs的电压非依赖性抑制。本项目的具体目的是：(1)确定哪些G蛋白与e18a偶联；(2)确定el8a如何参与D1型多巴胺受体(D1R)和血管紧张素II受体1型(AT1R)对N型通道的抑制；(3)确定el8a在介导多巴胺能神经元天然N型电流的G蛋白依赖性抑制中的作用。将在表达系统(tsA201细胞)和从小鼠中脑培养物分离的神经元中研究N型通道eL8a剪接变体，该培养物旨在表达DIR表达的神经元中的GFP。这些通道的功能将用电生理学和药理学来探索。G蛋白对克隆通道和天然通道的抑制作用将用GTPgS或GPCR激动剂进行评估。这种抑制将被(1)用siRNAs特异性地抑制包含e18a的N型通道，和(2)用抑制肽隔离e8a结合伙伴而被打破。总体而言，本项目旨在阐明N型通道的选择性剪接在控制对G蛋白介导的抑制的敏感性中的作用。相关性：帕金森氏病的神经退行性变特别影响中脑神经元。神经递质可以通过抑制钙通道来调节这些神经元的活动，钙通道是神经元之间交流的关键蛋白质。本研究项目旨在了解在中脑神经元中特异表达的钙通道的抑制作用。

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.