权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Biological significance of palytoxin-sensitive ion channel associated with NaィイD1+ィエD1,KィイD1+ィエD1-ATPase molecule

NaiiD1+IeD1、KiiD1+IeD1-ATPase分子相关的海藻毒素敏感离子通道的生物学意义

基本信息

批准号：
09460140
负责人：
ITO Katsuaki
金额：
$ 6.34万
依托单位：
University of Miyazaki
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

1) Electophysiological properties of palytoxin (PTX)-induced channel current in vascular smooth muscle cells and megakaryocytes were investigated using a patch clamp technique. PTX-induced channel in both cell types was permeable to monovalent cations but not to divalent cations. Ouabain, an inhibitor of Na,K-ATPase, suppressed the current, suggesting that PTX-sensitive channel is associated with Na,K-ATPase. A current dependent on Na pump was observed in the presence of PTX. This suggests that the Na pump is still operable after induction of the channel by PTX.2) PTX increased cytosolic CaィイD12+ィエD1 ([CaィイD12+ィエD1]ィイD2iィエD2) in vascular smooth muscle cells. Analysis of [CaィイD12+ィエD1]ィイD2iィエD2 mobilization revealed that one mechanism for the increase is activation of L-type CaィイD12+ィエD1 channels as a result of depolarization. Another mechanism is inhibition of CaィイD12+ィエD1 extrusion through NaィイD1+ィエD1-CaィイD12+ィエD1 exchanger since PTX increased [NaィイD1+ィエD1]ィイD2iィエD2 and caused depolar … More ization.3) To explore whether catalytic subunit of Na,K-ATPase is responsible for induction of PTX-sensitive channel, we transfected yeast cells, which do not have endogenous Na,K-ATPase, with wild type Na,K-ATPase (NNN) gene or its chimeric gene (NCN), in which the catalytic subunit was replaced with that of endoplasmic reticulum Ca-ATPase, and observed the effect of PTX on KィイD1+ィエD1 efflux. PTX increased KィイD1+ィエD1 efflux from NNN- and NCN-expressed cells but not from non-transformed cells. Ouabain inhibited the efflux. When ouabain-resistant NNN and NCN were transfected to yeast, PTX also increased KィイD1+ィエD1 efflux. KィイD1+ィエD1 efflux in these cells was insensitive to ouabain. Na azide and Na orthovanadate, which inhibit Na,K-ATPase, depressed the effect of PTX in an cDNA-transfected cells.These data suggest that PTX-sensitive channel is originally a pore to transport NaィイD1+ィエD1 and KィイD1+ィエD1 in the enzyme and the site of channel is far from the catalytic subunit. Induction of the channel by PTX seems to depend on a conformation state of the enzyme. Less

1)用膜片钳技术研究了海葵毒素（PTX）诱导的血管平滑肌细胞和巨核细胞通道电流的电生理特性。PTX诱导的通道在两种细胞类型是可渗透的一价阳离子，但不二价阳离子。Na，K-ATP酶抑制剂哇巴因（Ouabain）抑制该电流，提示PTX敏感通道与Na，K-ATP酶有关。在PTX存在下观察到依赖于Na泵的电流。2）PTX增加血管平滑肌细胞胞浆Ca ~（2+）D_1（[Ca ~（2+）D_1]~（2+）D_2）。对[Ca ~（2+）D_1]~（2+）D_2和~（2+）D_2动员的分析表明，增加的一种机制是由于去极化而激活L型Ca ~（2+）D_1通道。另一种机制是通过Na + D1+ Ca 2 + D1-Ca 2 + D1交换抑制Ca 2 + D12+ Ca 2 + D1的外排，因为PTX增加[Na + D1+ Ca 2 + D1] Ca 2 + D2 + Ca 2 + D1的外排， ...更多信息 3）为了探讨Na，K-ATP酶催化亚基是否参与了PTX敏感性通道的诱导，我们将野生型Na，K-ATP酶（NNN）基因或其嵌合基因（NCN）转染不含内源性Na，K-ATP酶的酵母细胞，观察PTX对K + ATP酶D1+ ATP酶D1流出的影响。PTX增加了NNN和NCN表达细胞的K β D1+ K β D1流出，但对非转化细胞无影响。哇巴因抑制外排。当哇巴因抗性NNN和NCN转染到酵母中时，PTX也增加了K β D1+ K β D1的流出。这些细胞中的K β D1+ K β D1流出对哇巴因不敏感。抑制Na，K-ATP酶活性的叠氮化钠和原钒酸钠可抑制PTX的作用，提示PTX敏感通道最初是一个转运Na ~+ ATP酶D1和K ~+ ATP酶D1的孔道，通道位置远离催化亚基。PTX诱导的通道似乎取决于酶的构象状态。少