Does alternative splicing regulate G protein inhibition of calcium channels?

描述（由申请人提供）：n型（CaV2.2）电压门控钙通道（vgcc）在几乎所有中枢和外周神经元的突触前末端表达，在那里它们控制触发胞外分泌的钙进入。因此，神经传递的效果与通过vgc的钙量直接相关。这种关系是利用经典形式的突触前调节：G蛋白介导的n型通道的抑制。控制G蛋白偶联受体（gpcr）与钙通道特异性偶联的分子规则尚不完全清楚。Lipscombe实验室最近的工作为通道-G蛋白相互作用特异性提供了一个令人兴奋和新颖的解释：CaV2.2前mrna的选择性剪接是控制G蛋白偶联到n型钙通道的分子机制。本研究的重点是另一个外显子el 8a在G蛋白介导的n型通道抑制中的作用。该建议的总体假设是：细胞特异性的el 8a包含使n型通道易受gpcr的电压无关抑制。该项目的具体目的是：(1)确定哪些G蛋白与e18a偶联；(2)确定el 8a如何参与D1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在多巴胺能神经元中介导G蛋白依赖性抑制天然n型电流中的作用。n型通道el 8a剪接变异体将在表达系统（tsA201细胞）和分离的小鼠中脑培养神经元中进行研究，以在表达dir1的神经元中表达GFP。利用电生理学和药理学方法探讨这些通道的功能。克隆和天然通道的G蛋白抑制作用将用GTPgS或GPCR激动剂进行评估。这种抑制将被(1)用sirna特异性抑制含有e18a的n型通道和(2)用抑制肽隔离el 8a结合伙伴所破坏。总的来说，该项目旨在阐明n型通道的选择性剪接在控制G蛋白介导的抑制易感性中的作用。相关性：帕金森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重要蛋白质。本研究旨在了解钙通道在中脑神经元特异性表达的抑制作用。

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.