Plant Cyclic Nucleotide Gated Channels: Functional Characterization Using Cloned Channels and Native Plant Membranes

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)的分子特征。CNGCs参与信号转导，也参与植物对阳离子的吸收。之前由美国国家科学基金会资助的这个项目的工作导致了对植物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性质的差异有关。动物CNGCs(人类的6个基因)都具有相似的孔结构(在离子选择性过滤器区域)，并非选择性地输送Ca、Na和K。植物CNGCs的情况更为复杂；20个拟南芥CNGCs被模拟为具有不同的离子选择性过滤器。CNGCs被鉴定为第一个克隆的植物通道，它传递钙(一种重要的细胞质信号分子)和钠(植物吸收是限制作物生长的主要因素)。其中一个通道具有孔结构，代表了一种迄今未知的分子范例，允许区分K/Na传导。以前的工作包括应用异源表达系统(卵母细胞、细胞培养和酵母突变体)来描述这些特征。新项目继续了这项工作，但扩大了对植物CNGCs的分析，以便产生关于植物CNGCs的阳离子传导特性如何与植物体内的养分吸收和移动有关的新信息。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制分析。这一成果为该项目提供了基础，包括利用天然膜表征植物天然CNGC蛋白复合体的电生理特性，以及在异源系统中表达的克隆植物CNGC的结构/功能研究。我们将对野生型细胞膜上的天然CNGC电流和缺乏特定CNGC基因的突变植物进行比较。这些天然CNGC的电生理分析将与CNGC依赖的阳离子在植物体内的吸收和移动的研究以及CNGC突变植物在不同营养液条件下生长时的表型分析一起进行。CNGC异构体特异性抗体将被用来产生关于天然植物CNGC亚基组成的新信息，并与表达谱一起，允许剖析单个CNGC如何对植物中的阳离子通量做出贡献。通过三维建模和功能域的定点突变，深入了解这些蛋白质的结构/功能关系。通过建模了解蛋白质的结构/功能是学生从分子水平上理解生物系统的重要基础。作为让高中生物教师接触到这些见解的持续推广计划的一部分，将开发一个教学实践，并在暑假期间为准备成为中学生物教师的本科生提供培训。每节训练课将招募10名学生，并向他们提供津贴，让他们参加为期两周的暑期实习，培训学生使用基于网络的蛋白质建模软件，并学习使用简单的生物系统(即培养酵母突变株以测试蛋白质功能)，从而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关注于“教授教师”的过程，以便接受高中生物教师培训的本科生能够参与到后基因组时代的下一个生物学领域；即了解蛋白质结构与生物功能的关系。

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.