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Physical dimensions of the pores of VDACL, VSOR and CFTR chloride channels as putative ATP-channels.

作为假定 ATP 通道的 VDACL、VSOR 和 CFTR 氯离子通道的孔的物理尺寸。

基本信息

批准号：
15590201
负责人：
SABIROV Ravshan
金额：
$ 2.3万
依托单位：
National Institute for Physiological Sciences (2004)Okazaki National Research Institutes (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Maxi-anion channel, volume-sensitive outwardly rectifying (VSOR) anion channel and cystic fibrosis transmembrane conductance regulator (CFTR) channel have been suggested to mediate an electrogenic transport of ATP by different groups. This function requires a pore wide enough for passing a bulky ATP molecule. The purpose of the present project was to evaluate the physical dimensions of the pores of the three putative ATP-channels. (1) Single maxi-anion channels were recorded from membrane patches excised from mouse mammary C127 cells. We first analyzed the permeability of the channel to a series of organic anions of different size. We found a linear relationship between relative permeability of organic anions of different size and their relative ionic mobility (measured as the ratio of ionic conductance) with a slope close to 1, suggesting that organic anions tested with radii up to 0.49 nm (lactobionate) move inside the channel by free diffusion. In the second approach, we, for the fi … More rst time, succeeded in pore sizing by the nonelectrolyte exclusion method in single-channel patch-clamp experiments. The cut-off radii of PEG molecules that could access the channel from intracellular (1.16 nm) and extracellular (1.42 nm) sides indicated an asymmetry of the two entrances to the channel pore. Measurements by symmetrical two-sided application of PEG molecules yielded an average functional pore radius of 〜1.3 nm. (2) Single VSOR channels were recorded from the cell-attached patches of human epithelial Intestine 407 cells pre-swollen in hypotonic Hi-K+ solutions. PEG 200-300 (Rh=0.27-0.53 nm) effectively suppressed the single-channel currents, whereas PEG 400-4000 (Rh=0.62-1.91 nm) had little or no effect. The cut-off radius of the VSOR channel pore was assessed to be 0.63 nm. (3) Single CFTR channels were recorded from cell-attached and excised membrane patches from HEK293T cells transiently transfected with CFTR gene using a bi-cistronic vector with GFP gene as a reporter. The effect of polyethylene glycols on the single-channel CFTR currents was different depending on whether they were added from extracellular or intracellular side. The cut-off radius of PEG molecules that could access the channel from the intracellular side (〜1.0 nm) was larger than that from the extracellular side (〜0.6 nm) indicating an asymmetry of the two channel entrances.The radius of ATP4- and MgATP2- (about 0.6-0.7 nm) is close to the limiting size of the VSOR channel pore, and narrowest part of CFTR channel. The size of maxi-anion channel (〜1.3 nm) is largest among these three channels; it is best suited to its function as an ATP channel and, where present, serves as the preferred pathway for release of ATP4- and/or MgATP2-. Less

最大阴离子通道、体积敏感外整流（VSOR）阴离子通道和囊性纤维化跨膜电导调节剂（CFTR）阴离子通道被认为通过不同的基团介导ATP的电致转运。这个功能需要一个足够宽的孔来通过一个大体积的ATP分子。本项目的目的是评估三个假定的atp通道的孔隙的物理尺寸。(1)从小鼠乳腺C127细胞切除的膜斑块中记录到单个最大阴离子通道。我们首先分析了该通道对一系列不同大小的有机阴离子的渗透性。我们发现，不同尺寸的有机阴离子的相对渗透率与其相对离子迁移率（以离子电导率测量）之间呈线性关系，斜率接近1，表明半径达0.49 nm的有机阴离子（乳酸盐）通过自由扩散在通道内移动。在第二种方法中，我们首次在单通道膜片钳实验中成功地采用非电解质排除法进行了孔径的测定。可以从细胞内（1.16 nm）和细胞外（1.42 nm）两侧进入通道的PEG分子的截止半径表明通道孔的两个入口不对称。对称两侧应用PEG分子的测量结果显示，平均功能孔半径约为1.3 nm。(2)在低压Hi-K+溶液中预肿胀的人上皮性肠407细胞贴壁斑块中记录到单个VSOR通道。PEG 200-300 （Rh=0.27-0.53 nm）有效抑制单通道电流，而PEG 400-4000 （Rh=0.62-1.91 nm）几乎没有作用。VSOR通道孔的截止半径评估为0.63 nm。(3)利用以GFP基因为报告基因的双顺反子载体，从瞬时转染CFTR基因的HEK293T细胞贴壁和切除的膜斑块上记录单个CFTR通道。聚乙二醇对单通道CFTR电流的影响取决于它们是从细胞外还是细胞内添加的。从细胞内进入通道的PEG分子的截止半径（~ 1.0 nm）大于从细胞外进入通道的截止半径（~ 0.6 nm），表明两个通道入口不对称。ATP4-和MgATP2-的半径（约0.6 ~ 0.7 nm）接近VSOR通道孔的极限尺寸，CFTR通道的最窄部分。最大阴离子通道尺寸最大（~ 1.3 nm）；它最适合其作为ATP通道的功能，并且在存在的情况下，作为释放ATP4-和/或MgATP2-的首选途径。少

项目成果

期刊论文数量（59）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

P.D.Bell, J.-Y.Lapointe, R.Z.Sabirov, S.Hayashi, J.Peti-Peterdi, K.Manabe, G.Kovacs, Y.Okada: "Macula densa cell signaling involves ATP release through a maxi anion channel."Proc.Natl.Acad.Sci.USA. 100. 4322-4327 (2003)

P.D.Bell、J.-Y.Lapointe、R.Z.Sabirov、S.Hayashi、J.Peti-Peterdi、K.Manabe、G.Kovacs、Y.Okada：“致密斑细胞信号传导涉及通过 maxi 阴离子通道释放 ATP。”

DOI：
发表时间：
期刊：
影响因子：
0
作者：
通讯作者：

Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischemic or hypoxic conditions.

ATP 传导阴离子通道在缺血或缺氧条件下新生大鼠心肌细胞 ATP 释放中的作用。

DOI：
发表时间：
2004
期刊：
J.Physiol.(London) 559
影响因子：
0
作者：
Lee E-J.;Iai H.;Koizumi N.;Sano H.;Kanzaki-Kato N. et al.;Harada M. et al.;Kameda T. et al.;Yan M.Y.et al.;Ise N.et al.;Nakayama K. et al.;A.K.Dutta
通讯作者：
A.K.Dutta

Ischemia-induced enhancement of CFTR expression on the plasma membrane in neomatal rat ventricular myocytes.

缺血诱导的新生大鼠心室肌细胞质膜上 CFTR 表达增强。