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

描述（由申请人提供）：钙活化的氯离子通道（CaCC）在真核生物中广泛表达，但其分子身份在二十多年来一直是个谜。2008年，我们的研究小组和另外两个小组得出了同样的结论，即TMEM16A形成了CaCC。我们的研究进一步表明TMEM16B也形成CaCC。CaCC作为TMEM16“功能未知的跨膜蛋白”家族的两个成员的分子鉴定使我们能够解决有关CaCC如何工作以及CaCC如何参与大脑神经元信号传导的问题。我们建议探讨以下问题：钙如何激活CaCC？发现TMEM16A-CaCC可被多种二价阳离子激活，但对显性负突变型钙调蛋白（CaM）表达或抗CaM抗体（可减少TRPV1通道快速反应）的应用不敏感，我们对酸性残基进行了系统的诱变调查，以了解它们可能参与钙结合或门控。为此，我们首先证明了果蝇TMEM16家族成员形成CaCC。然后，我们诱变了几十个高度保守的酸性残基，以确定对钙门控重要的酸性残基。通过改变这些位置的侧链，并检查突变通道对不同大小和化学性质的二价阳离子的敏感性，我们将验证这些酸性残基的一个子集配位钙，而其他残基可能提供与二价阳离子相互作用的第二壳羧酸盐的假设。这项研究还可能确定参与钙门控转导的残基。CaCC如何与渗透离子相互作用以促进氯离子渗透？在发现TMEM16F形成一个小电导钙激活的非选择性阳离子通道（SCAN）之后，我们寻找TMEM16A-CaCC和TMEM16F-SCAN之间不同的氨基酸，并发现在两个单独的跨膜（TM）片段中两个残基的突变会改变离子的选择性。此外，我们与约翰霍普金斯大学的李敏博士合作，完成了30万化合物的高通量筛选，以鉴定新的CaCC阻滞剂。我们将在诱变研究中使用新的CaCC孔阻滞剂和硫醇试剂来识别和表征潜在的孔衬里残留物。CaCC是如何参与神经元信号的钙调节的？我们发现TMEM16B在大脑中广泛表达，而不是TMEM16A。为了产生TMEM16B敲除小鼠，我们敲除了作为TMEM16B表达报告基因的法酰化mCherry的编码序列。在发现TMEM16B在下橄榄高表达与小脑共济失调、肌张力障碍、特发性震颤和其他疾病如胎儿酒精综合征有关后，我们将检测TMEM16B- cacc是否参与下橄榄神经元的尖峰波形和阈下振荡及其调节。

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.