Isoform Specific Ubiquitination of the N-type Calcium Channel

N 型钙通道的亚型特异性泛素化

• 批准号: 7809338
• 负责人: Spiro Anthony Marangoudakis
• 金额: $ 2.73万
• 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 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7809338
• 关键词: Adverse effects; Affect; Amino Acids; Biological Assay; Calcium Channel; Cells; Code; Essential Amino Acids; Exons; G-Protein-Coupled Receptors; Gel; Goals; Immunoprecipitation; Lead; Mediating; Mus; N-Type Calcium Channels; Neurons; Nociception; Nociceptors; Pathway interactions; Pattern; Peptides; Protein Isoforms; Public Health; RNA Splicing; Regulation; Relative (related person); Research; Running; Screening procedure; Spinal Ganglia; Stretching; Surface; Testing; Transgenic Mice; Ubiquitin; Ubiquitination; Western Blotting; Work; Yeasts; chronic pain; density; in vivo; interest; mutant; novel; research study; voltage; yeast two hybrid system

DESCRIPTION (provided by applicant): The long-term aim of this project is to better understand the ubiquitination and regulation of Cav2.2e[37a] and Cav2.2e[37b]- two splice isoforms of the Cav2.2 N-type channel. These two isoforms differ in only fourteen amino acid residues, but have been shown to be ubiquitinated at different levels. These two isoforms also undergo an interesting interplay in the nociceptive neurons of the dorsal root ganglia (DRG). All neurons contain Cav2.2e[37b], however Cav2.2e[37a] has very limited expression, and is enriched in nociceptive neurons of the DRG. Since the Cav2.2 channel is already one of the most efficacious and specific targets to treat chronic pain, understanding the regulation of these two types of channels is likely to aid in understanding and treating chronic pain mediated through nociceptive neurons. The research proposed here aims to determine the mechanisms involved in regulating Cav2.2e[37a] and Cav2.2e[37b] function through ubiquitination. This proposal has three aims. Aim one will utilize novel mouse lines that express only one isoform of Cav2.2- Cav2.2e[37a] or Cav2.2e[37b], to determine ubiquitination levels of each isoform in vivo. Immunoprecipitation (IP) of the channel will be performed from these mouse lines and run on SDS-PAGE gel. Afterward a western blot will be performed where ubiquitin and Cav2.2 are probed. Ubiquitination levels of the two isoforms will be compared. The second aim will utilize mutants of ubiquitin to determine the type of ubiquitin attachment to the two channel isoforms. These experiments will utilize at least three mutants of ubiquitin (KOR, K48R, and K63R) that are deficient in forming ubiquitin chains. The mutants will be expressed in primary DRG neuronal cultures of the mice proposed to be used in aim one. The channel will then be immunoprecipitated, and probed for ubiquitin to determine if either isoform is modified with ubiquitin chains differentially. For aim three, mutants of the channel will be created in which stretches of amino acids from Cav2.2e[37a] will be replaced with those from Cav2.2e[37b], and vice versa, to determine which of the fourteen amino acids are responsible for ubiquitination of Cav2.2e[37b]. After this aim is completed, point mutants of Cav2.2 may also be created to determine the exact amino acid residue(s) responsible for the differential ubiquitination. This work is relevant to public health because it will lead to better understanding of the pathways underlying the degradation and modulation of N-type calcium channels. If these pathways can be better understood, then treatments for chronic pain can likely be created that are more potent and cause fewer side-effects than those that currently exist.

描述(由申请人提供)：该项目的长期目标是更好地了解Cav2.2e[37a]和Cav2.2e[37b]-Cav2.2N型通道的两种剪接异构体-的泛素化和调节。这两种异构体只有14个氨基酸残基不同，但已被证明在不同的水平上泛素化。这两种异构体在背根神经节(DRG)的伤害性感受神经元中也经历了有趣的相互作用。所有神经元都含有Cav2.2e[37b]，但Cav2.2e[37a]的表达非常有限，并且富含背根节的伤害性神经元。由于Cav2.2通道已经是治疗慢性疼痛最有效和最特异的靶点之一，了解这两种类型通道的调节可能有助于理解和治疗由伤害性神经元介导的慢性疼痛。本研究旨在确定通过泛素化调节Cav2.2e[37a]和Cav2.2e[37b]功能的机制。这项提议有三个目标。目的利用仅表达一种Cav2.2-Cav2.2e[37a]或Cav2.2e[37b]亚型的新型小鼠品系，测定各亚型在体内的泛素化水平。将从这些小鼠系中进行通道的免疫沉淀(IP)，并在SDS-PAGE凝胶上运行。之后，将进行蛋白质印迹，检测泛素和Cav2.2。我们将比较两种异构体的泛素化水平。第二个目标将利用泛素的突变体来确定泛素与两种通道异构体的结合类型。这些实验将利用至少三种泛素突变体(KOR、K48R和K63R)，它们在形成泛素链方面存在缺陷。这些突变体将在原代培养的小鼠背根神经节神经元中表达，该小鼠被提议用于Aim One。然后，该通道将被免疫沉淀，并探测泛素，以确定是否有两种不同的异构体被不同的泛素链修饰。出于第三个目的，将创建通道的突变体，其中来自Cav2.2e[37a]的氨基酸片段将被Cav2.2e[37b]的氨基酸替代，反之亦然，以确定14个氨基酸中的哪一个负责Cav2.2e[37b]的泛素化。在完成这一目标后，还可以创建Cav2.2的点突变来确定负责差异泛素化的确切氨基酸残基(S)。这项工作与公众健康相关，因为它将有助于更好地理解N型钙通道降解和调节的潜在途径。如果能够更好地了解这些途径，那么治疗慢性疼痛的方法可能会比目前存在的更有效、副作用更少的治疗方法。