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) 使用膜片钳技术研究了血管平滑肌细胞和巨核细胞中海藻毒素 (PTX) 诱导的通道电流的电生理特性，两种细胞类型中 PTX 诱导的通道可渗透单价阳离子，但不能渗透二价阳离子。 Na,K-ATPase 抑制剂抑制了电流，表明 PTX 敏感通道与 Na,K-ATPase 相关，观察到电流依赖于 Na 泵。 PTX 的存在表明 Na 泵在 PTX 诱导通道后仍然可操作。2) PTX 增加血管平滑肌细胞中的胞质 CaD12+D1 ([CaD12+D1]D2iD2)。 ]iiD2iieD2 动员表明增加的一种机制是激活 L 型由于 PTX 增加 [Nai D1+D1]D2iD2 并导致去极化，另一种机制是通过 NaD1+D1-CaD12+D1 交换器抑制 CaiD12+D1 通道。3) 探讨是否催化亚基Na,K-ATPase 负责诱导 PTX 敏感通道，我们用野生型 Na,K-ATPase (NNN) 基因或其嵌合基因 (NCN) 转染不具有内源性 Na,K-ATPase 的酵母细胞，其中催化亚基被内质网Ca-ATP酶的催化亚基取代，并观察到PTX对KiD1+D1流出量增加的影响。当哇巴因抗性NNN和NCN转染酵母时，来自NNN和NCN表达细胞的K-D1+D1流出抑制了流出，PTX也增加了K-D1+D1 D1流出。这些细胞中的 D1+D1 流出对叠氮化钠和 Na 不敏感。原钒酸盐可抑制Na,K-ATPase，从而抑制cDNA转染细胞中PTX的作用。这些数据表明，PTX敏感通道最初是在酶中转运Na,K-ATPase和K-D1+ D1的孔，并且PTX 的通道位点远离催化亚基，似乎取决于酶的构象状态。