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Structure and function of the SLAC/SLAH anion channel family

SLAC/SLAH阴离子通道家族的结构和功能

基本信息

批准号：
137502288
负责人：
Professor Dr. Rainer Hedrich
金额：
--
依托单位：
Lehrstuhl für Botanik I: Molekulare Pflanzenphysiologie und Biophysik
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2009
资助国家：
德国
起止时间：
2008-12-31 至 2013-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/137502288?language=en
关键词：
Structure function SLAC SLAH anion

项目摘要

At the beginning of 2008 SLAC1, the first member of a small gene family in Arabidopsis, was identified. Lack of SLAC1 function prevented activation of guard cell anion channels. Since SLAC1 could not be functionally analysed yet in any heterologous expression system, the structure-function relationship of SLAC/SLAH family members remains unclear. The applicants recently succeeded to functionally express members of the SLAC/SLAH family. Based on the predicted primary structure of SLAC1 we analysed the membrane spanning domains and cytoplasmic N- and C-termini by using topology-sensitive reporters. Using molecular- and biochemical approaches the number of subunits required for channel function will be determined. Within the SLAC/SLAH anion channel family we identified two subtypes differing in anion permeability, susceptibility toward blockers, and gating. Via chimera between these subtypes and mutational analysis we will identify channel domains responsible for ion selectivity and voltage control. Based on functional studies with a set of SLAC/SLAH mutants combined with biochemical analysis with recombinant channel protein, a molecular model of the channel complex will be generated. The proposed working model will be validated and refined by testing model predictions via mutation analyses. Based on the model and high-throughput screens with a chemical library we will search for anion channel blockers of high-affinity and specificity. Taking advantage of the model-predicted channel’s 3D structure on one side and blocker chemistry on another, designed channel effectors will be generated. The latter will serve structure-function studies and represent new tools for testing anion channel physiology and may constitute the basis for new agrochemicals.

在2008年初，SLAC 1，拟南芥中的小基因家族的第一个成员，被确定。SLAC 1功能的缺乏阻止了保卫细胞阴离子通道的激活。由于SLAC 1在任何异源表达系统中都不能进行功能分析，因此SLAC/SLAH家族成员的结构-功能关系仍不清楚。申请人最近成功地功能性表达了SLAC/SLAH家族的成员。基于预测的SLAC 1的一级结构，我们分析了跨膜结构域和细胞质的N-和C-末端通过使用拓扑敏感的报告。使用分子和生物化学方法，将确定通道功能所需的亚基数量。在SLAC/SLAH阴离子通道家族中，我们确定了两种在阴离子渗透性、对阻滞剂的敏感性和门控方面不同的亚型。通过这些亚型之间的嵌合体和突变分析，我们将确定负责离子选择性和电压控制的通道结构域。基于一组SLAC/SLAH突变体的功能研究，结合重组通道蛋白的生化分析，将产生通道复合物的分子模型。拟议的工作模型将通过突变分析测试模型预测进行验证和改进。基于该模型和高通量筛选的化学库，我们将寻找高亲和力和特异性的阴离子通道阻断剂。利用模型预测的通道的一侧的3D结构和另一侧的阻断剂化学，将生成设计的通道效应器。后者将服务于结构-功能研究，代表测试阴离子通道生理学的新工具，并可能构成新农药的基础。