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Functional and molecular identification of ATP releasing channel

ATP释放通道的功能和分子鉴定

基本信息

批准号：
13670051
负责人：
SABIROV Ravshan
金额：
$ 2.24万
依托单位：
Okazaki National Research Institutes, National Institute for Physiological Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670051/
关键词：
ATP channel Anion channel Cell volume ATP release GFTR

项目摘要

In mouse C127 cells, we found a new swelling-activated Gd-sensitive anionic current, which had no outward rectification, and inactivated at voltages greater than ±25 mV. Single-channels of 300-400 pS in on cell and inside-out patches displayed inactivation pattern similar to whole-cell currents. The channels had an ATP-binding site in the middle of the pore and were ATP-conductive with P_<ATP>/P_<Cl> of 0.09. The pharmacological profile of single-channel activity was same to that found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel (VDACL) serves as a conductive pathway for the swelling-induced ATP release in C127i cells.Arachidonic acid (AA) down-regulated both VDACL currents and swelling-induced ATP release in physiological concentration range with K_d of 4-6 μM. This effect was direct and not mediated by downstream metabolic products. A membrane-impermeable analog arachidonyl coenzyme A was … More effective only from the cytosolic side suggesting that the binding site is localized intracellularly. ATP^4-currents were also reversibly inhibited by AA. Thus, we conclude that swelling-induced ATP release and its putative pathway, VDACL anion channel, are under a negative control by intracellular arachidonic acid signaling in mammary C 127 cells.We have attempted to identify VDACL channel in two different ways. (1) Since electrophysiologically, VDACL - channel resembles the mitochondrial voltage-dependent anion channel (VDAC), we have tested the hypothesis that VDACL corresponds to plasmalemmal (pl-) subtype of VDAC. We detected the mRNA expression of pl-VDACL which was identical to the reported pl-VDAC sequence. However, we failed to reproduce VDACL channels phenotype by heterologous expression. Next, we tested the effects specific antagonists of mitochondrial ATP/ADP translocator, atracyloside and bongcrekic acid on ATP release and found no consistent effect, therefore, this transporter is unlikely to be a molecular candidate for VDACL channel. Less

在小鼠 C127 细胞中，我们发现了一种新的肿胀激活的 Gd 敏感阴离子电流，该电流没有外向整流，并且在电压大于 ±25 mV 时失活。细胞上和内向外斑块中 300-400 pS 的单通道显示出类似于全细胞电流的失活模式。该通道在孔的中部有一个 ATP 结合位点，并且具有 ATP 传导性，P_<ATP>/P_<Cl> 为 0.09。单通道活性的药理学特征与肿胀诱导的 ATP 释放中发现的相同。因此，可以得出结论，体积和电压依赖性ATP传导大电导阴离子通道（VDACL）是C127i细胞中肿胀诱导的ATP释放的传导途径。花生四烯酸（AA）在K_d为4-6 μM的生理浓度范围内下调VDACL电流和肿胀诱导的ATP释放。这种影响是直接的，不是由下游代谢产物介导的。膜不可渗透的类似物花生四烯酰辅酶 A 仅在胞质侧有效，表明结合位点位于细胞内。 AA 也可逆地抑制 ATP^4 电流。因此，我们得出结论，肿胀诱导的 ATP 释放及其假定的途径 VDACL 阴离子通道受到乳腺 C 127 细胞中细胞内花生四烯酸信号传导的负控制。我们尝试以两种不同的方式鉴定 VDACL 通道。 (1)由于在电生理学上，VDACL-通道类似于线粒体电压依赖性阴离子通道(VDAC)，因此我们检验了VDACL对应于VDAC的质膜(pl-)亚型的假设。我们检测到pl-VDACL的mRNA表达，其与报道的pl-VDAC序列相同。然而，我们未能通过异源表达重现 VDACL 通道表型。接下来，我们测试了线粒体 ATP/ADP 转运蛋白、atracyloside 和 bongcrekic Acid 的特异性拮抗剂对 ATP 释放的影响，没有发现一致的效果，因此，该转运蛋白不太可能是 VDACL 通道的分子候选者。较少的