Plant Cyclic Nucleotide Gated Ion Channels; Structure: Function Analysis of a Newly Identified and Unique Family of Proteins

本实验室最近克隆并首次进行了拟南芥环核苷酸(CNMP)门控钾离子通道(‘AtCNGC2’)的功能研究。环核苷酸门控(非选择性)阳离子通道(CNGC‘s)(在动物中)是配基门控的，传导K、Na和Ca，并受细胞内钙、钙调蛋白和环核苷酸水平的调节。CNGC通常在信号转导途径中发挥作用，提供了一种机制，外部信号感知通过该机制调用信号级联，进而改变细胞功能。CAMP和cGMP在动物特定的信号转导系统中的作用是众所周知的。然而，它们在植物特定信号转导系统中的作用还不是很清楚。AtCNGC2显然是植物中一个大的基因家族的成员：数据库搜索已经在拟南芥基因组中发现了10个编码CNGC的序列。这些植物CNGC的推导氨基酸序列在功能上与动物的CNGC序列不同。这种植物类离子通道的电生理特性尚未阐明。AtCNGC2和其他植物CNGC将在能够进行膜片/电压钳分析的异源系统(非洲爪哇卵母细胞和/或HEK293细胞培养)中表达。一些AtCNGC2同源物将在卵母细胞中表达，用于功能鉴定。这项工作的主要目的将是分析atCNGC2及其同系物的电生理结构-功能，首先关注孔的选择性过滤，其次是这些通道的环核苷酸结合域。这些研究将包括AtCNGC2的定点突变，然后对表达突变通道的卵母细胞进行电压钳分析。将详细分析这些通道的离子选择性分布的差异，以及对cAMP和cGMP亲和力的差异。这一研究目标应该允许比较这种植物通道家族成员的初级蛋白质结构的差异如何与功能和调控的差异相关联。随着这个大基因家族的其他成员被克隆，将进行以编码序列为探针的Northern分析，以监测该基因家族不同成员在拟南芥中的表达模式。这项研究项目将产生关于离子通道表征最深入研究领域之一的新信息：离子选择性的分子基础。其他人的一项里程碑式的研究导致了对K选择性离子通道的第一次X射线结晶学分析。这项开创性的工作证实，K通道的孔选择性过滤器中的三重氨基酸(‘GYG’)是通道电导有利于K而不是Na的绝对必要条件。对AtCNGC2孔选择性过滤器的模拟表明，氨基酸三联体‘and’与这一独特的通道传导K并针对Na进行特异性选择的能力有关，表明AtCNGC2以一种迄今在生物学上未知的方式选择K而不是Na的电导！这一发现表明，目前对通道离子选择性分布的分子基础的理解应该重新评估。该项目的意义得到以下支持：a)该通道家族在功能域上的初级序列差异表明了不同的通道特性；b)大量克隆的植物通道用于分子水平的研究是史无前例的；c)通道特性(例如K和Ca电导，而不是Na)可能是一个独特的生物学范例，可能为了解蛋白质结构如何影响通道特性提供有价值的见解；d)这个蛋白质家族需要cAMP和cGMP来激活，这些研究可能为环核苷酸如何调节植物细胞中的离子通道提供洞察力。

This laboratory has recently cloned and undertaken the first functional characterization of Arabidopsis thaliana cyclic nucleotide (cNMP) gated K-conducting ion channel ('AtCNGC2'). Cyclic nucleotide gated (nonselective) cation channels (cngc's) (in animals) are ligand-gated, conduct K, Na, and Ca, and are regulated by cytosolic levels of Ca, calmodulin, and cyclic nucleotides. Cngc's typically function in signal transduction pathways, providing a mechanism by which external signal perception invokes signaling cascades that in turn alter cellular functions. The role that cAMP and cGMP play in specific signal transduction systems in animals is well known. However, their involvement in specific signal transduction systems in plants is not well understood. AtCNGC2 is apparently a member of a large gene family in plants: Database searches have identified 10 sequences in the A. thaliana genome which encode putative cngc's. The deduced amino acid sequences of these plant cngc's differ from the animal cngc sequences in regions that are critical for function. The electrophysiological properties of this plant class of ion channels have not been elucidated. AtCNGC2 and other plant cngc's will be expressed in heterologous systems amenable to patch/voltage clamp analysis (Xenopus laevis oocytes, and/or HEK293 cell cultures). Some of the AtCNGC2 homologs will be expressed in oocytes for functional characterization. The primary objective of this work will be an electrophysiological structure-function analysis of AtCNGC2 and homologs, focusing firstly on the selectivity filter of the pore and secondly on the cyclic nucleotide binding domains of these channels. These studies will include site-directed mutagenesis of AtCNGC2, followed by voltage clamp analysis of oocytes expressing the mutated channels. A detailed analysis will be undertaken of differences in ion selectivity profiles of these channels, and also differences in affinity for cAMP and cGMP. This research objective should allow for a comparison of how differences in the primary protein structure of members of this plant channel family correlate with differences in function and regulation. As other members of this large gene family are cloned, Northern analyses using coding sequences as a probe will be undertaken to monitor expression patterns of different members of this gene family in Arabidopsis. This research project will generate new information about one of the most intensively studied areas of ion channel characterization: The molecular basis of ion selectivity. A landmark study by others led to the first X-ray crystallographic analysis of a K-selective ion channel. This pioneering work confirmed that a triplet of amino acids ('GYG') in the pore selectivity filter of K channels is absolutely required for channel conductance to favor K over Na. Modeling of the AtCNGC2 pore selectivity filter indicates that the amino acid triplet 'AND' is associated with this unique channel's ability to conduct K and specifically select against Na, indicating that AtCNGC2 selects for K over Na conductance in a manner heretofore unknown in biology! This finding suggests that current understanding of the molecular basis for ion selectivity profiles of channels should be reappraised. The significance of this project is supported by the following: a) the primary sequence differences amongst this channel family in functional domains suggests different channel properties; b) the availability of a large family of cloned plant channels for study at the molecular level is unprecedented; c) the channel properties (e.g. K and Ca conductance, but not Na) may be a unique biological paradigm that may provide valuable insights into how protein architecture affects channel properties; and d) this family of proteins requires cAMP and cGMP for activation and these studies may provide insights into how cyclic nucleotides regulate ion channels in plant cells.