TMEM16A Channel Stoichiometry and Subunit Interaction

描述（由申请人提供）：钙激活的氯离子通道（CaCC）于20世纪80年代初首次在蝾螈视网膜中被观察到，此后被发现与各种现象有关，从嗅觉感觉神经元的信号转导到上皮中的液体分泌。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。尽管其重要性，编码CaCC的基因直到2008年才被发现，当时三个实验室独立发现了产生CaCC电流的TMEM16A克隆。在这一发现的基础上，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，开始对该通道进行初步的基本生物物理表征。初步数据表明，TMEM16A与其他TMEM16A分子特异性共免疫沉淀，表明在CaCC复合体中存在不止一个亚基。为了确定亚基的确切数量，TMEM16A将连接到两个不同的标签并异源表达。使用电生理学、免疫沉淀和生物素化，将观察到只含有一个标签而不含有另一个标签的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，它们就越不可能完全相同——并由生物分布建模。为了验证该方法的结果，将测定天然和化学交联条件下含有tmem16a的CaCC配合物的表观分子量。CaCC配合物的分子量应该是单个TMEM16A亚基质量的整数倍。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点中荧光团漂白事件的数量应等于存在的TMEM16A亚基的数量。本研究计划的第二组实验将确定负责亚基寡聚化的蛋白质结构域。系统突变将在TMEM16A的开放阅读框中进行，共同免疫沉淀和电生理实验将确定哪些突变会破坏亚基相互作用。根据这些数据，将TMEM16F （TMEM16家族成员，通常不与TMEM16A共免疫沉淀）中的同源结构域替换为TMEM16A序列，以确定这些序列是否足以使嵌合蛋白与野生型TMEM16A共免疫沉淀。

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. PUBLIC HEALTH RELEVANCE: The goal of this project is to examine the structure of the mammalian calcium-activated chloride channel. This channel is considered a possible target for the treatment of hypertension, cystic fibrosis, and cancer. As there are currently no specific small-molecule blockers nor activators of this channel, my research will contribute to drug design that will find molecules to efficiently target this ion channel in these diseases.