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: 8008809
• 负责人: Spiro Anthony Marangoudakis
• 金额: $ 1.18万
• 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 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8008809
• 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）：本项目的长期目标是更好地了解Cav2.2e[37 a]和Cav2.2e[37 b]的泛素化和调控-Cav2.2 N型通道的两种剪接异构体。这两种异构体仅在14个氨基酸残基上不同，但已显示在不同水平上被泛素化。这两种亚型在背根神经节（DRG）的伤害感受神经元中也发生有趣的相互作用。所有神经元都含有Cav2.2e[37 b]，然而Cav2.2e[37 a]的表达非常有限，并且在DRG的伤害感受神经元中富集。由于Cav2.2通道已经是治疗慢性疼痛的最有效和特异性靶点之一，因此了解这两种类型通道的调节可能有助于理解和治疗通过伤害性神经元介导的慢性疼痛。本文提出的研究旨在确定通过泛素化调节Cav2.2e[37 a]和Cav2.2e[37 b]功能的机制。这项建议有三个目的。目的是利用仅表达Cav2.2- Cav2.2e[37 a]或Cav2.2e[37 b]的一种同种型的新小鼠系，以确定体内每种同种型的泛素化水平。将对这些小鼠系进行通道免疫沉淀（IP），并在SDS-PAGE凝胶上运行。然后进行蛋白质印迹，其中探测泛素和Cav2.2。将比较两种亚型的泛素化水平。第二个目标是利用泛素的突变体来确定泛素与两种通道亚型的连接类型。这些实验将利用至少三种在形成泛素链方面有缺陷的泛素突变体（KOR、K48 R和K63 R）。突变体将在拟用于目的一的小鼠的原代DRG神经元培养物中表达。然后对通道进行免疫沉淀，并探测泛素，以确定任一亚型是否被泛素链差异修饰。对于目标三，将创建通道的突变体，其中来自Cav2.2e[37 a]的氨基酸段将被来自Cav2.2e[37 b]的氨基酸段替换，反之亦然，以确定十四个氨基酸中的哪一个负责Cav2.2e的泛素化[37 b]。在此目标完成后，还可以产生Cav2.2的点突变体以确定负责差异泛素化的确切氨基酸残基。这项工作与公共卫生有关，因为它将导致更好地了解N型钙通道降解和调节的潜在途径。如果能够更好地理解这些途径，那么就有可能创造出比目前存在的更有效、副作用更少的慢性疼痛治疗方法。