Molecular Analysis of Inward Rectifier K^+channels in CNS

CNS 内向整流 K^ 通道的分子分析

基本信息

批准号：
07044262
负责人：
KURACHI Yoshihisa
金额：
$ 9.47万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044262/
关键词：
K^+ channels brain cloning knockout synaps GTP-binding protein polyamine cluster 中枢神経系分子生物学

项目摘要

To analyze inwardly rectifying potassium channels (IRKs) in the brain, we have cloned cDNAs of GIRK1B and GIRK2B.These channels may participate in the reguration of GTP-binding protein-regulated potassium channels (K_G). We also cloned SUR2B (sulfonyl urea receptor 2B) cDNA which is a subunit of the ATP-regulated potassium channel expressed in vascular smooth muscle. Expression of SUR2B was ubiquitous and was also found in the brain. The genes for IRK3, K_<AB>-2, and GIRK1 were isolated. These genes will be used for knockout studies.To investigate the distribution of IRLs in the central nervous system, we performed in situ hybridization. These IRK channels were specifically expressed in special sets of neurons. Large amount of IRK2 mRNA was detected in the granular cell later of cerebellum, on the other hand, IRK3 mRNA was localized in the forebrain. Immunohistochemical studies showed that GIRK1 was expressed in presynapses in the paraventricular nubleus. We have previously demonstrate … More d that K_<AB>-2 was predominantly expressed in glial cells. Using anti-K_<AB>-2 antibody, we further analized the distribution of K_<AB>-2, and found that K_<AB>-2 was expressed in Muller cells, retinal glial cells, and marginal cells, which are thought to contribute elevation of endolymph potential of inner ear. In these cells, K_<AB>-2 may have a key role inthe transport of K^+ from cells to cells. We also found that K_<AB>-2 was clustered in the membrane of Muller cells. K_<AB>-2 was associated with PSD-95/SAP90 and clustered when K_<AB>-2 was co-expressed with PSD-95/SAP90 in HEK293T cells. We also found that teh expression of PSD-95/SAP90 family proteins enhanced tha K_<AB>-2 current in HEK293T cells. Electrophysiological properties of inwardly rectifying potassium channels were studied to investigate the function and regulation of these channels in the brain. We found that inward rectification of K_G channels and cloned IRK2 channels was caused by intracellular polyamines. We also found that the binding of more than two molecules (could be four) of GTP-binding protein betagamma heterodimer to a single K_G channel was needed to open the channel. As a K_G channel was composed of four subunits of GIRK channels, our data suggested that each subunit binds one molecule of betagamma heterodimer. Less

为了分析大脑中内部整流的钾通道（IRK），我们有GIRK1B和GIRK2B的CDNA。这些通道可能参与GTP结合蛋白调节的钾通道（K_G）的调节。我们还克隆SUR2B（磺酰基尿素受体2B）cDNA，这是在血管平滑肌中表达的ATP调节钾通道的亚基。 SUR2B的表达无处不在，也发现在大脑中。分离了IRK3，K_ <ab> -2和GIRK1的基因。这些基因将用于敲除研究。为了研究中枢神经系统中IRL的分布，我们进行了原位杂交。这些愤怒的通道是在特殊的神经元中特异性表达的。另一方面，在小脑的颗粒细胞中检测到大量IRK2 mRNA，而IRK3 mRNA位于前脑。免疫组织化学研究表明，GIRK1在旁牙的刺中表达。我们以前已经证明了……更多的d表明K_ <ab> -2主要在神经胶质细胞中表达。使用抗K_ <ab> -2抗体，我们进一步分析了K_ <ab> -2的分布，并发现K_ <ab> -2在Muller细胞，残留神经胶质细胞和边缘细胞中表达，这些细胞被认为促进了内耳呼吸的内淋巴电位的升高。在这些细胞中，k_ <ab> -2可能在从细胞到细胞的k^+转运中具有关键作用。我们还发现K_ <ab> -2聚集在Muller细胞的膜中。 K_ <ab> -2与PSD-95/SAP90相关联，当K_ <ab> -2与HEK293T单元格中的PSD-95/SAP90共表达时，将聚类并聚集。我们还发现，PSD-95/SAP90家族蛋白的TEH表达增强了HEK293T细胞中的Tha K_ <ab> -2电流。研究了内部整流钾通道的电生理特性，以研究这些通道在大脑中的功能和调节。我们发现，K_G通道和克隆IRK2通道的内向整流是由细胞内多胺引起的。我们还发现，需要两个以上的GTP结合蛋白Betagamma Heterodimer与单个K_G通道的结合以打开通道。由于K_G通道由GIRK通道的四个亚基组成，因此我们的数据表明每个亚基结合了一个betagamma杂质二聚体的一个分子。较少的