Plant Cyclic Nucleotide Gated Channels: Functional Characterization Using Cloned Channels and Native Plant Membranes
植物环核苷酸门控通道:使用克隆通道和天然植物膜进行功能表征
基本信息
- 批准号:0344141
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:Continuing Grant
- 财政年份:2004
- 资助国家:美国
- 起止时间:2004-04-15 至 2008-03-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
This project focuses on the molecular characterization of plant cyclic nucleotide gated nonselective cation channels (CNGCs). CNGCs act in signal transduction cascades, and also are involved in cation uptake into plants. Prior NSF-funded work on this project led to the first electrophysiological analyses of plant CNGCs, focusing on how their molecular architecture is related to differences in their ion conductance properties. Animal CNGCs (six genes in humans) all have similar pore architectures (in the ion selectivity filter region) and nonselectively conduct Ca, Na, as well as K. The situation with plant CNGCs is more complex; the 20 Arabidopsis CNGCs were modeled to have different ion selectivity filters. CNGCs were identified as the first cloned plant channels that conduct Ca (an important cytosolic signaling molecule) and Na (uptake into plants is a major limitation to crop growth). One of the channels was shown to have a pore architecture representing a heretofore unknown molecular paradigm allowing for discrimination between K/Na conduction. Prior work involved the application of heterologous expression systems (oocytes, cell cultures, and yeast mutants) for these characterizations. The new project continues this work, but expands the analysis of plant CNGCs so as to generate new information about how the cation conduction properties of plant CNGCs are related to nutrient uptake and movement within plants. Work underlying the project has led to the first voltage clamp analyses of CNGC currents in native plant membranes. This accomplishment provides a basis for the project to include studies using native membranes to characterize the electrophysiological properties of native CNGC protein complexes in plants, as well as structure/function studies of cloned plant CNGCs expressed in heterologous systems. Comparisons will be made between native CNGC currents in membranes of wild type, and mutant plants that lack specific CNGC genes. These electrophysiological analyses of native CNGCs will be undertaken along with studies of CNGC-dependent cation uptake and movement within plants, and analysis of CNGC mutant plant phenotypes when grown under various nutrient solution regimes. CNGC isoform-specific antibodies will be employed to generate new information about the subunit composition of native plant CNGCs and, along with expression profiling, allow for the dissection of how individual CNGCs contribute to cation fluxes in plants.Insight into the structure/function relationships of these proteins has been aided by three-dimensional modeling and site-directed mutagenesis of functional domains. Understanding protein structure/function through modeling is an important basis for students to understand biological systems at the molecular level. As part of a continuing outreach program to expose high school biology teachers to these insights, a teaching practicum will be developed and offered during summer semester breaks to undergraduate students who are preparing to be secondary school biology teachers. For each training session, ten of these students will be recruited and provided with stipends to participate in the two-week summer practicum that will train students to access web-based protein modeling software, and learn to work with simple biological systems (i.e. growth of yeast mutants to test protein function) so as to bring this information into the high school biology classroom. The broader impact of this project is the focus on a process of 'teaching the teachers' so that undergraduate students training as high school biology teachers can participate in the next horizon of biology in this post-genomic era; i.e. to gain an understanding of how protein structure is related to biological function.
本项目主要研究植物环核苷酸门控非选择性阳离子通道(CNGC)的分子特性。CNGC在信号转导级联中起作用,并且还参与阳离子吸收到植物中。之前NSF资助的关于该项目的工作导致了植物CNGC的第一次电生理分析,重点是它们的分子结构如何与它们的离子电导特性差异相关。动物CNGC(人类中的六个基因)都具有相似的孔结构(在离子选择性过滤区),并且非选择性地传导Ca、Na和K。植物CNGC的情况更复杂; 20个拟南芥CNGC被建模为具有不同的离子选择性过滤器。CNGC被鉴定为第一个克隆的传导Ca(一种重要的胞质信号分子)和Na(植物吸收是作物生长的主要限制)的植物通道。其中一个通道被证明具有代表迄今未知的分子范式的孔结构,允许区分K/Na传导。先前的工作涉及应用异源表达系统(卵母细胞,细胞培养物和酵母突变体)进行这些表征。新项目继续这项工作,但扩展了植物CNGC的分析,以产生关于植物CNGC的阳离子传导特性如何与植物内营养吸收和运动相关的新信息。该项目的基础工作导致了对天然植物膜中CNGC电流的第一次电压钳分析。这一成就为该项目提供了基础,包括使用天然膜来表征植物中天然CNGC蛋白复合物的电生理特性的研究,以及在异源系统中表达的克隆植物CNGC的结构/功能研究。将在野生型膜中的天然CNGC电流与缺乏特定CNGC基因的突变植物之间进行比较。天然CNGC的这些电生理学分析将与植物内CNGC依赖性阳离子吸收和运动的研究一起沿着进行,并且当在各种营养液方案下生长时,分析CNGC突变体植物表型。CNGC异构体特异性抗体将产生新的信息,天然植物CNGC的亚基组成,沿着表达谱,允许解剖如何个人CNGC有助于阳离子fluxinplants.Insight到这些蛋白质的结构/功能关系的三维建模和功能结构域的定点诱变。通过建模理解蛋白质结构/功能是学生在分子水平上理解生物系统的重要基础。作为一个持续的外展计划,让高中生物教师接触到这些见解的一部分,将开发一个教学实习,并在夏季学期休息期间提供给准备成为中学生物教师的本科生。 对于每一个培训课程,这些学生中的十个将被招募并提供助学金参加为期两周的夏季实习,将培训学生访问基于网络的蛋白质建模软件,并学习使用简单的生物系统(即酵母突变体的生长来测试蛋白质功能),以便将这些信息带入高中生物课堂。这个项目的更广泛的影响是集中在一个过程中的“教学教师”,使本科生培训高中生物学教师可以参与生物学的下一个视野在这个后基因组时代,即获得蛋白质结构的理解是如何与生物功能。
项目成果
期刊论文数量(0)
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Wolf-Dieter Reiter其他文献
Positively supercoiled DNA in a virus-like particle of an archaebacterium
古细菌病毒样颗粒中的正超螺旋 DNA
- DOI:
10.1038/321256a0 - 发表时间:
1986-05-15 - 期刊:
- 影响因子:48.500
- 作者:
Marc Nadal;Gilles Mirambeau;Patrick Forterre;Wolf-Dieter Reiter;Michel Duguet - 通讯作者:
Michel Duguet
Wolf-Dieter Reiter的其他文献
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{{ truncateString('Wolf-Dieter Reiter', 18)}}的其他基金
Function and Control of Xyloglucan Galactosylation in Arabidopsis
拟南芥中木葡聚糖半乳糖基化的功能和控制
- 批准号:
0725940 - 财政年份:2007
- 资助金额:
-- - 项目类别:
Standard Grant
Plant Calcium Conducting Channels: Linking Molecular Architecture to Roles in Innate Immunity Signal Transduction
植物钙传导通道:将分子结构与先天免疫信号转导中的作用联系起来
- 批准号:
0721679 - 财政年份:2007
- 资助金额:
-- - 项目类别:
Standard Grant
Characterization of Xyloglucan Galactosyltransferase Homologs in Arabidopsis
拟南芥中木葡聚糖半乳糖基转移酶同系物的表征
- 批准号:
0215535 - 财政年份:2002
- 资助金额:
-- - 项目类别:
Continuing Grant
Genetic Analysis of Plant Cell Wall Matrix Components
植物细胞壁基质成分的遗传分析
- 批准号:
9728779 - 财政年份:1998
- 资助金额:
-- - 项目类别:
Standard Grant
Characterization of Higher Plant Phosphoinositide-specific Phospholipase C
高等植物磷酸肌醇特异性磷脂酶 C 的表征
- 批准号:
9728024 - 财政年份:1998
- 资助金额:
-- - 项目类别:
Standard Grant
Characterization of Arabidopsis Mutants Altered in Fucose- Containing Cell Wall Polysaccharides
含岩藻糖细胞壁多糖改变的拟南芥突变体的表征
- 批准号:
9405739 - 财政年份:1994
- 资助金额:
-- - 项目类别:
Continuing Grant
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