An Oocyte Expression/Patch Clamp Analysis System for the Study of Membrane Channel Proteins

罗格斯大学/UMDNJ的几个研究小组专注于动物和植物系统中新发现的膜蛋白（通道，受体，转运体）的分子特征。利用分子遗传学方法克隆了这些膜蛋白编码多肽成分的假定cdna。在不久的将来，表征这些cdna的研究项目的快速发展将取决于表征由cdna翻译产物促进的转运功能的能力。一种最先进的技术可以用来促进这种表征是卵母细胞表达/膜片钳分析。这涉及到将这些cdna转录的cRNA微量注射到青蛙卵母细胞中，导致卵母细胞膜中转运蛋白的翻译和功能性表达。通过使用贴片/电压钳设备监测表达这些蛋白的卵母细胞质膜上的离子电流，然后可以用来研究这些基因产物的特定运输功能。该提案寻求资金在罗格斯大学建立第一个卵母细胞表达/膜片钳分析系统。本提案中描述的研究项目都将利用该设备来深入了解目标膜蛋白的结构/功能关系。具体的研究目标，将被研究人员阅读使用拟议的设备如下。1)迄今为止不知道存在于植物中的长通道基因将被表征。具体来说，将研究编码植物K+通道亚基的cdna，细胞内植物膜上的配体11型（30-40 kD多肽）K+通道，以及具有独特孔隙序列（以前在植物或动物膜中未知）的K+通道，该通道可能是植物Na+摄取途径的候选国。2)对参与植物细胞壁胼胝质（纤维素）生物合成的酶复合物的多肽组分进行表征。编码这些多肽的cdna与MIP（主要内在蛋白）或水通道蛋白基因家族的成员具有序列同源性。3)对谷氨酸（Glu）兴奋性氨基酸受体家族中一个新成员的编码cDNA进行了鉴定。谷氨酸受体介导哺乳动物中枢神经系统的快速兴奋性神经传递。glur是一种阳离子通道，被称为嗜离子受体。克隆的cDNA编码一种独特的剪接变体，它是GluR的截断受体形式。4. 核编码蛋白通过跨越细胞器双膜的特定通道进入植物叶绿体。六种叶绿体包膜蛋白的复合体已被确定为叶绿体蛋白进口机制的组成部分。编码其中一种蛋白质IAP75的cDNA已被克隆。序列分析表明，IAP75与细菌外膜孔蛋白等通道蛋白具有二级结构相似性。我们将对IAP75的类孔蛋白性质进行表征。

Several research groups at Rutgers/UMDNJ have focused on the molecular characterization of newly-identified membrane proteins (channels, receptors, transporters) in animal and plant systems. Use of molecular genetic approaches has led to the cloning of putative cDNAs encoding polypeptide components of these membrane proteins. Rapid advancement of the research programs characterizing these cDNAs will, in the near future, depend on the capability to characterize the transport functions facilitated by the translation products of the cDNAs. One state-of-the-art technology which can be employed to facilitate this characterization is oocyte expression/patch clamp analysis. This involves the microinjection of cRNA transcribed from these cDNAs into frog (Xenopus laevis) oocytes, leading to translation and functional expression of the transport proteins in oocyte membranes. Monitoring of ion currents across the plasmalemma of oocytes expressing these proteins by use of patch/voltage clamp equipment can then be employed to study the specific transport functions of these gene products. This proposal seeks funds to set up the first oocyte expression/patch clamp analysis system at Rutgers. The research programs described in this proposal will all make use of this equipment to gain insights into the structure/function relationships of the target membrane proteins. The specific research objectives which will be perused by investigators using the proposed equipment are as follows. 1 ) lon channel genes heretofore not known to be present in plants will be characterized. Specifically, cDNAs encoding a subunit of plant K+ channels, a ligandgated type 11 (30-40 kD polypeptide) K+ channel native to an intracellular plant membrane, and a K+ channel which has a unique pore sequence (unknown previously in plant or animal membranes) and is a likely candidate for the Na+ uptake pathway in plants will be studied. 2) Polypeptide components of the enzyme complex involved in plant ce ll wall callose (cellulose) biosynthesis will be characterized. The cDNAs encoding these polypeptides share sequence homology with members of the MIP (major intrinsic protein) or aquaporin gene family. 3) A cDNA encoding a novel member of the family of glutamate (Glu) family of excitatory amino acid receptors will be characterized. Glu receptors (GluRs) mediate rapid excitatory neurotransmission in the mammalian central nervous system. GluRs are a type of cation channel known as ionotropic receptors. The cloned cDNA encodes a unique splice variant that is a truncated receptor form of a GluR. 4. Nuclear-encoded proteins are imported into plant cell chloroplasts via specific channels spanning the double membrane delimiting the organelle. A complex of six chloroplast envelope membrane proteins have been identified as components of the chloroplast protein import machinery. A cDNA encoding one of these proteins, IAP75, has been cloned. Sequence analysis of IAP75 indicates that it has secondary structure similarity to channel proteins such as bacterial outer membrane porins. The porinlike nature of IAP75 will be characterized.