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

最大阴离子通道、容量敏感外向整流(VSOR)阴离子通道和囊性纤维化跨膜电导调节(CFTR)通道被认为在不同的群体中介导了ATP的生电转运。这一功能需要一个足够宽的孔来通过体积庞大的ATP分子。本项目的目的是评估三种假定的ATP通道的毛孔的物理尺寸。(1)从小鼠乳腺C127细胞膜片上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。我们发现不同大小的有机阴离子的相对渗透率与它们的相对离子迁移率(以离子电导之比衡量)呈线性关系，斜率接近1，这表明被测的有机阴离子(乳糖酸盐)在通道内以自由扩散的方式移动。在第二种方法中，我们为FI…在单通道膜片钳实验中，首次用非电解质排除法成功地确定了微孔的大小。从胞内(1.16 nm)和胞外(1.42 nm)侧进入通道的聚乙二醇单分子的截断半径表明通道孔的两个入口是不对称的。通过对称双面施加聚乙二醇分子的测量，得到了平均功能孔半径约1.3 nm。(2)在低渗Hi-K溶液中预膨胀的人上皮细胞407细胞贴壁斑块可记录到单个VSOR通道。聚乙二醇200-300(Rh=0.27-0.53 nm)可有效抑制单通道电流，而聚乙二醇400-4000(Rh=0.62-1.91 nm)对单通道电流几乎无影响。VSOR通道孔的截止半径为0.63 nm。(3)以绿色荧光蛋白基因为报告基因的双顺反子载体，从瞬时转导CFTR基因的HEK293T细胞贴壁和摘除的膜片上记录到单个CFTR通道。聚乙二醇对单通道CFTR电流的影响取决于它们是从胞外还是胞内添加的。从胞内侧进入通道的聚乙二醇单分子的截止半径(~1.0 nm)大于从胞外侧进入通道的截止点半径(~0.6 nm)，表明两个通道入口的不对称性，ATP4-和MgATP2-的半径(约0.6-0.7 nm)接近VSOR通道孔的极限尺寸，是CFTR通道的最窄部分。最大阴离子通道(~1.3 nm)是这三种通道中最大的；它最适合作为ATP通道的功能，并且在存在的情况下，是ATP4-和/或MgATP2-释放的首选途径。较少

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