TMEM16A Channel Stoichiometry and Subunit Interaction

TMEM16A 通道化学计量和亚基相互作用

基本信息

批准号：
8382856
负责人：
Jason Tien
金额：
$ 4.22万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8382856
关键词：
Ablation Afferent Neurons Binding Binomial Distribution Biotinylation CLCA2 gene Calcium Cardiac Myocytes Cells Chimera organism Chimeric Proteins Chloride Channels Chloride Ion Chlorides Co-Immunoprecipitations Complex Cystic Fibrosis Data Disease Drug Design Electrodes Electrophysiology (science)Epithelium Event Family member Fluids and Secretions Frequencies Genes Goals Immunoprecipitation Individual Ion Channel Laboratories Link Malignant Neoplasms Measures Mediating Membrane Modeling Molecular Structure Molecular Weight Mutate Mutation Neurons Oocytes Open Reading Frames Peripheral Photobleaching Population Proteins Regulation Research Retina Salamander Series Signal Transduction Smooth Muscle Structure Tertiary Protein Structure Total Internal Reflection Fluorescent Translations Weight airway epithelium cell type crosslink fluorophore hypertension treatment intestinal epithelium kidney cell research study small molecule stoichiometry voltage clamp

项目摘要

DESCRIPTION (provided by applicant): The calcium-activated chloride channel (CaCC) was first observed in salamander retina in the early 1980s and has since then been found to be responsible for diverse phenomena ranging from signal transduction in the olfactory sensory neuron to fluid secretion in the epithelium. In disease, the CaCC has been considered a target for the treatment of hypertension, cystic fibrosis, and cancer. Despite its importance, the gene encoding the CaCC was not identified until 2008 when three laboratories independently discovered a TMEM16A clone that produced the CaCC current. Building on this discovery, the project described in this research plan will begin the initial basic biophysical characterization of this channel by determining the number of TMEM16A subunits that oligomerize to form a functional CaCC. Preliminary data indicate that TMEM16A co-immunoprecipitates specifically with other TMEM16A molecules, suggesting that more than one subunit is present in the CaCC complex. To determine the exact number of subunits, TMEM16A will be linked to two different tags and heterologously expressed. Using electrophysiology, immunoprecipitation, and biotinylation, the proportion of CaCC complexes that contain only subunits with one tag but not the other will be observed. This proportion is a function of the number of subunits required to assemble a channel - the more subunits required, the less likely that they are all the same - and is modeled by the bionomial distribution. To validate the results from this approach, the apparent molecular weight of TMEM16A-containing CaCC complexes under native and chemically-crosslinked conditions will be determined. The molecular weight of a CaCC complex should be some integer multiple of the weight of individual TMEM16A subunits. Lastly, GFP-tagged TMEM16A will be expressed in oocytes and photobleached under TIRF microscopy. The number of fluorophore bleaching events in a single CaCC punctum should equal the number of TMEM16A subunits present. A second set of experiments in this research plan will identify the protein domains responsible for subunit oligomerization. Systematic mutations will be made in TMEM16A's open reading frame and co- immunopreciptation and electrophysiological experiments will determine which mutations abolish subunit interactions. From this data, homologous domains in TMEM16F (a TMEM16 family member that does not normally co-immunoprecipitate with TMEM16A) will be replaced with TMEM16A sequences to determine whether these sequences are sufficient for chimeric proteins to co-immunoprecipitate with wildtype TMEM16A.

描述（由申请人提供）：钙激活氯离子通道 (CaCC) 于 20 世纪 80 年代初首次在蝾螈视网膜中观察到，此后被发现负责从嗅觉感觉神经元中的信号转导到液体分泌等多种现象在上皮细胞中。在疾病方面，CaCC 被认为是治疗高血压、囊性纤维化和癌症的靶点。尽管它很重要，但直到 2008 年，三个实验室独立发现了产生 CaCC 电流的 TMEM16A 克隆，编码 CaCC 的基因才被发现。基于这一发现，本研究计划中描述的项目将通过确定寡聚形成功能性 CaCC 的 TMEM16A 亚基的数量来开始对该通道的初步基本生物物理表征。初步数据表明，TMEM16A 与其他 TMEM16A 分子发生特异性共免疫沉淀，表明 CaCC 复合物中存在多个亚基。为了确定亚基的确切数量，TMEM16A 将连接到两个不同的标签并进行异源表达。使用电生理学、免疫沉淀和生物素化，将观察仅包含带有一个标签但不包含另一个标签的亚基的 CaCC 复合物的比例。该比例是组装通道所需的子单元数量的函数 - 所需的子单元越多，它们全部相同的可能性就越小 - 并且通过生物项分布进行建模。为了验证该方法的结果，将确定包含 TMEM16A 的 CaCC 复合物在天然和化学交联条件下的表观分子量。 CaCC 复合物的分子量应该是单个 TMEM16A 亚基重量的整数倍。最后，GFP 标记的 TMEM16A 将在卵母细胞中表达，并在 TIRF 显微镜下进行光漂白。单个 CaCC 泪点中荧光团漂白事件的数量应等于存在的 TMEM16A 亚基的数量。该研究计划中的第二组实验将确定负责亚基寡聚化的蛋白质结构域。将在 TMEM16A 的开放阅读框架中进行系统突变，并且免疫共沉淀和电生理学实验将确定哪些突变消除了亚基相互作用。根据该数据，TMEM16F（通常不与 TMEM16A 进行免疫共沉淀的 TMEM16 家族成员）中的同源结构域将被 TMEM16A 序列替换，以确定这些序列是否足以让嵌合蛋白与野生型 TMEM16A 进行免疫共沉淀。