Isoform Specific Ubiquitination of the N-type Calcium Channel

项目描述（由申请人提供）：本项目的长期目标是更好地了解Cav2.2 n型通道的两种剪接异构体Cav2.2e[37a]和Cav2.2e[37b]的泛素化和调控。这两种异构体仅在14个氨基酸残基上有所不同，但已被证明在不同水平上泛素化。这两种异构体在背根神经节（DRG）的伤害性神经元中也经历了有趣的相互作用。所有神经元均含有cav2.2 .2e[37b]，但cav2.2 .2e[37a]表达非常有限，且在DRG的伤害性神经元中富集。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，了解这两种通道的调控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控cav2.2 .2e[37a]和cav2.2 .2e[37b]功能的机制。这项提议有三个目的。目的一将利用仅表达Cav2.2- Cav2.2e[37a]或Cav2.2e[37b]一种异构体的新型小鼠系来测定体内各异构体的泛素化水平。通道的免疫沉淀（IP）将在这些小鼠系上进行，并在SDS-PAGE凝胶上运行。随后进行western blot，检测泛素和Cav2.2。将比较两种异构体的泛素化水平。第二个目标将利用泛素突变体来确定泛素附着在两个通道亚型上的类型。这些实验将利用至少三种缺乏形成泛素链的泛素突变体（KOR， K48R和K63R）。该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，以确定泛素链是否被不同的同种异构体修饰。对于目标三，将创建通道的突变体，其中Cav2.2e[37a]的氨基酸片段将被Cav2.2e[37b]的氨基酸片段所取代，反之亦然，以确定14种氨基酸中哪一种负责Cav2.2e[37b]的泛素化。在此目标完成后，还可以创建Cav2.2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，因为它将使人们更好地了解n型钙通道降解和调节的途径。如果能更好地理解这些途径，那么就有可能创造出比现有疗法更有效、副作用更少的慢性疼痛疗法。

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.