Molecular, genetic & physiological studies of calcium-activated chloride channels

分子、遗传

• 批准号: 8694437
• 负责人: LILY Y JAN
• 金额: $ 34.56万
• 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: 2011-07-15 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8694437
• 关键词: Action Potentials; Afferent Neurons; Amino Acids; Antibodies; Ataxia; Binding Sites; Brain; Brain Stem; Brain region; Calcium; Calcium Binding; Calcium Channel; Calmodulin; Cations; Cells; Cerebellar Ataxia; Cerebellum; Chemistry; Chimeric Proteins; Chloride Channels; Chloride Ion; Code; Collaborations; Collection; Cytoplasmic Tail; Disease; Divalent Cations; Dominant-Negative Mutation; Drosophila genus; Dystonia; Elderly; Essential Tremor; Eukaryota; Family; Family member; Feedback; Fetal Alcohol Syndrome; Fiber; Generations; Genes; Grant; Green Algae; Habenula; Homologous Gene; Inferior; Integral Membrane Protein; Ion Channel; Ions; Knock-in Mouse; Knockout Mice; Lateral Septal Nucleus; Learning; Medial; Mediating; Membrane; Membrane Potentials; Molecular; Molecular Genetics; Mutagenesis; Mutate; N-Methyl-D-Aspartate Receptors; Nervous system structure; Neurons; Olives - dietary; Pattern; Peripheral; Physiological; Positioning Attribute; Process; Reagent; Regulation; Reporter; Role; Sensory; Side; Signal Transduction; Sleeplessness; Slice; Spinal Ganglia; Structure; Structure of trigeminal ganglion; Sulfhydryl Reagents; Surveys; Synapses; System; TRPV1 gene; Tachyphylaxis; Testing; Touch sensation; United States National Institutes of Health; Whole-Cell Recordings; Work; carboxylate; channel blockers; high throughput screening; hippocampal pyramidal neuron; interest; member; mutant; novel; public health relevance; response; serotonin receptor; small hairpin RNA

DESCRIPTION (provided by applicant): Calcium-activated chloride channels (CaCC) are broadly expressed in eukaryotes but their molecular identity remained enigmatic for over a score of years. In 2008 our group and two other groups reached the same conclusion that TMEM16A forms CaCC. Our study further showed that TMEM16B also forms CaCC. Molecular identification of CaCC as two members of the TMEM16 family of "transmembrane proteins with unknown function" has enabled us to approach questions concerning how CaCC works and how CaCC contributes to neuronal signaling in the brain. We propose to approach the following questions: How does calcium activate CaCC? Having found that TMEM16A-CaCC can be activated by a variety of divalent cations but is insensitive to dominant negative mutant calmodulin (CaM) expression or application of anti-CaM antibody that can reduce TRPV1 channel tachyphylaxis, we took on the task for a systematic mutagenesis survey of acidic residues for their possible involvement in calcium binding or gating. To this end, we first showed that a Drosophila TMEM16 family member forms CaCC. We then mutagenized several dozens of highly conserved acidic residues to identify acidic residues important for calcium gating. By varying the side chain at these positions and examining the mutant channel sensitivity to divalent cations of different size and chemistry, we will test the hypothesis that a subset of thes acidic residues coordinates calcium whereas others may provide second-shell carboxylates that interact with divalent cations. This study may also identify residues involved in the transduction of calcium gating. How does CaCC interact with permeant ions to facilitate chloride ion permeation? Having made the surprise finding that TMEM16F forms a small-conductance calcium-activated non-selective cation channel (SCAN), we looked for amino acids that are different between TMEM16A-CaCC and TMEM16F-SCAN, and found that mutagenesis of two residues in two separate transmembrane (TM) segments alter ion selectivity. Moreover, in collaboration with Dr. Min Li at Johns Hopkins, we have finished high throughput screening of >300,000 compounds to identify novel CaCC blockers. We will use novel CaCC pore blockers and thiol reagents in our mutagenesis studies to identify and characterize potential pore-lining residues. How might CaCC be involved in the calcium modulation of neuronal signaling? We found broad expression of TMEM16B but not TMEM16A in the brain. To generate TMEM16B knockout mice, we knocked in the coding sequence for farnesylated mCherry as a reporter for TMEM16B expression. Having found high expression of TMEM16B in the inferior olive implicated in cerebellar ataxia, dystonia, essential tremor and other disorders such as fetal alcohol syndrome, we will test for TMEM16B-CaCC involvement in the spike waveform and subthreshold oscillation and their modulation in inferior olive neurons.

描述（由申请人提供）：钙活化的氯化物通道（CACC）在真核生物中广泛表达，但它们的分子身份在多年以上一直保持着神秘状态。 2008年，我们的小组和其他两个小组得出了与TMEM16A形成CACC相同的结论。我们的研究进一步表明，TMEM16B也形成了CACC。 CACC的分子鉴定是“具有未知功能的跨膜蛋白” TMEM16家族的两个成员，这使我们能够解决有关CACC如何工作的问题以及CACC如何促进大脑中的神经元信号传导。我们建议解决以下问题：钙如何激活CACC？ Having found that TMEM16A-CaCC can be activated by a variety of divalent cations but is insensitive to dominant negative mutant calmodulin (CaM) expression or application of anti-CaM antibody that can reduce TRPV1 channel tachyphylaxis, we took on the task for a systematic mutagenesis survey of acidic residues for their possible involvement in calcium binding or gating.为此，我们首先表明果蝇TMEM16家庭成员形成CACC。然后，我们诱变了几十个高度保守的酸性残基，以鉴定酸性残基对钙门控重要。通过在这些位置上改变侧链，并检查突变通道对不同大小和化学的二价阳离子的敏感性，我们将测试以下假设：这些假设是，酸性残基的子集坐标钙，而其他人可能会提供与分离阳离子相互作用的第二壳羧酸盐。这项研究还可以鉴定出涉及钙门转导的残基。 CACC如何与渗透离子相互作用以促进氯离子渗透？在发现TMEM16F形成小导钙激活的非选择性阳离子通道（SCAN）的令人惊讶的发现之后，我们寻找的氨基酸在TMEM16A-CACC和TMEM16F-SCAN之间有不同的氨基酸，并发现两个单独的跨膜（TM）seplecteries sealterivity selectivity selectivity selectivity selectivity termbrane seplectivity the tmem16a-cacc和tmem16f-scan。此外，与约翰·霍普金斯（Johns Hopkins）的米·李（Min Li）博士合作，我们已经完成了> 300,000种化合物的高吞吐量筛选，以识别新颖的CACC阻滞剂。我们将在我们的诱变研究中使用新型的CACC孔隙阻滞剂和硫醇试剂来识别和表征潜在的孔隙残基。 CACC如何参与神经元信号的钙调节？我们发现大脑中TMEM16B但TMEM16A的广泛表达。为了生成TMEM16B敲除小鼠，我们以Farneylated Mcherry作为TMEM16B表达的记者的编码序列进行了敲门。在发现与小脑共济失调，肌张力障碍，基本震颤和其他疾病（如胎儿酒精综合征）有关的下橄榄中，TMEM16B的表达很高后，我们将测试TMEM16B-CACC在峰值波形和亚thes骨振荡中的TMEM16B-CACC介入，并在下质橄榄神经元中进行调节。