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

描述（由申请人提供）：钙激活氯离子通道（CaCC）在真核生物中广泛表达，但其分子身份在几十年内仍然是个谜。在2008年，我们的团队和另外两个团队得出了相同的结论，即TMEM 16 A形成CaCC。我们的研究进一步表明，TMEM 16 B也形成CaCC。CaCC作为TMEM 16家族的两个成员的分子鉴定“具有未知功能的跨膜蛋白”使我们能够接近有关CaCC如何工作以及CaCC如何有助于大脑中的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？已经发现TMEM 16 A-CaCC可以被多种二价阳离子激活，但对显性负突变体钙调蛋白（CaM）表达或可以降低TRPV 1通道快速耐受的抗CaM抗体的应用不敏感，我们承担了酸性残基的系统诱变调查的任务，以确定它们可能参与钙结合或门控。为此，我们首先表明果蝇TMEM 16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，并检查突变体通道对不同大小和化学性质的二价阳离子的敏感性，我们将测试以下假设：这些酸性残基的一个子集与钙配位，而其他的则可能提供与二价阳离子相互作用的第二壳羧酸盐。这项研究还可能确定参与钙门控转导的残基。CaCO 3如何与渗透离子相互作用以促进氯离子渗透？在令人惊讶地发现TMEM 16 F形成小电导钙激活的非选择性阳离子通道（SCAN）之后，我们寻找TMEM 16 A-CaCC和TMEM 16 F-SCAN之间不同的氨基酸，并发现两个单独的跨膜（TM）区段中的两个残基的诱变改变离子选择性。此外，我们与约翰霍普金斯的Min Li博士合作，完成了对> 300，000种化合物的高通量筛选，以鉴定新型CaCC阻断剂。我们将在我们的诱变研究中使用新型CaCC孔阻断剂和硫醇试剂来鉴定和表征潜在的孔衬残基。CaCC如何参与神经元信号的钙调节？我们发现TMEM 16 B在脑中广泛表达，但TMEM 16 A没有。为了产生TMEM 16 B敲除小鼠，我们敲入法尼基化mCherry的编码序列作为TMEM 16 B表达的报告基因。已经发现下橄榄核中TMEM 16 B的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，我们将测试TMEM 16 B-CaCC参与下橄榄核神经元中的尖峰波形和阈下振荡及其调制。

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.