TMEM16A Channel Stoichiometry and Subunit Interaction

TMEM16A 通道化学计量和亚基相互作用

• 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
• 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

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，此后发现其负责从嗅觉感觉神经元中的信号转导到上皮中的液体分泌的多种现象。在疾病中，CaCC被认为是治疗高血压、囊性纤维化和癌症的靶点。尽管其重要性，编码CaCC的基因直到2008年才被确定，当时三个实验室独立地发现了产生CaCC电流的TMEM 16 A克隆。基于这一发现，本研究计划中描述的项目将通过确定寡聚化形成功能性CaCC的TMEM 16 A亚基的数量来开始该通道的初始基本生物物理表征。初步数据表明，TMEM 16 A与其他TMEM 16 A分子特异性共免疫沉淀，表明CaCC复合物中存在不止一个亚基。为了确定亚基的确切数目，TMEM 16 A将与两个不同的标签连接并异源表达。使用电生理学、免疫沉淀和生物素化，将观察仅含有具有一个标签而不含有另一个标签的亚基的CaCC复合物的比例。这个比例是组装通道所需的亚基数量的函数-所需的亚基越多，它们完全相同的可能性就越小-并且由生物分布建模。为了验证来自该方法的结果，将确定在天然和化学交联条件下含TMEM 16 A的CaCC复合物的表观分子量。CaCC复合物的分子量应该是单个TMEM 16 A亚基重量的某个整数倍。最后，GFP标记的TMEM 16 A将在卵母细胞中表达并在TIRF显微镜下光漂白。单个CaCC点中荧光团漂白事件的数目应等于存在的TMEM 16 A亚基的数目。本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TMEM 16 A的开放阅读框中进行系统突变，并且共免疫沉淀和电生理学实验将确定哪些突变消除亚基相互作用。根据该数据，TMEM 16 F（通常不与TMEM 16 A共免疫沉淀的TMEM 16家族成员）中的同源结构域将被TMEM 16 A序列取代，以确定这些序列是否足以使嵌合蛋白与野生型TMEM 16 A共免疫沉淀。