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

Rutgers/UMDNJ的几个研究小组专注于动物和植物系统中新鉴定的膜蛋白（通道，受体，转运蛋白）的分子特征。分子遗传学方法的使用已经导致克隆了编码这些膜蛋白的多肽组分的推定cDNA。在不久的将来，表征这些cDNA的研究计划的快速进展将取决于表征由cDNA的翻译产物促进的运输功能的能力。可用于促进这种表征的一种最先进的技术是卵母细胞表达/膜片钳分析。这涉及将从这些cDNA转录的cRNA显微注射到青蛙（非洲爪蟾）卵母细胞中，导致卵母细胞膜中转运蛋白的翻译和功能表达。通过使用膜片钳/电压钳设备监测跨表达这些蛋白质的卵母细胞质膜的离子电流，然后可以用于研究这些基因产物的特异性转运功能。该提案寻求资金，以建立罗格斯大学的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计划都将利用该设备来深入了解靶膜蛋白的结构/功能关系。研究者将使用拟议设备仔细研究的具体研究目标如下。1）迄今为止不知道存在于植物中的离子通道基因将被表征。具体地，编码一种 将研究植物K+通道的亚基、天然存在于细胞内植物膜的配体门控的11型（30-40 kD多肽）K+通道以及具有独特孔序列（先前在植物或动物膜中未知）并且是植物中Na+吸收途径的可能候选者的K+通道。2)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将被表征。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共享序列同源性。3)将表征编码兴奋性氨基酸受体的谷氨酸（Glu）家族的新成员的cDNA。谷氨酸受体（GluRs）介导哺乳动物中枢神经系统中的快速兴奋性神经传递。 GluR是一种称为离子型受体的阳离子通道。克隆的cDNA编码独特的剪接变体，其是GluR的截短受体形式。4.核编码的蛋白质通过跨越界定细胞器的双膜的特定通道输入植物细胞叶绿体。一个由六种叶绿体被膜蛋白组成的复合体被鉴定为叶绿体蛋白输入机制的组成部分。编码这些蛋白质之一，IAP 75的cDNA已被克隆。IAP 75的序列分析表明，它与细菌外膜孔蛋白等通道蛋白具有二级结构相似性。将表征IAP 75的孔蛋白样性质。

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.