Functional and molecular identification of ATP releasing channel

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，在大于±25 mV的电压下失活。300-400pS的单通道内细胞和内向外的贴片显示出类似于全细胞电流的失活模式。该通道在孔道中央有一个ATP结合部位，具有ATP导电性，P&lt；ATP&gt；/P&lt；Cl&gt；为0.09。单通道活性的药理曲线与肿胀诱导的ATP释放的药理曲线相同。花生四烯酸(AA)在生理浓度范围内(Kd为4~6μM)下调VDACL电流和膨胀性大电导阴离子通道的释放，这种作用不是由下游代谢产物直接介导的。不透膜的花生四烯基辅酶A类似物…仅在胞浆侧更有效，这表明结合部位定位于细胞内。AA对ATP^4-电流也有可逆性抑制作用。因此，我们认为，肿胀诱导的ATP释放及其可能的途径，即VDACL阴离子通道，在乳腺C127细胞中受到细胞内花生四烯酸信号的负调控。我们试图通过两种不同的方法来鉴定VDACL通道。(1)由于VDACL-通道在电生理上类似于线粒体电压依赖性阴离子通道(VDAC)，我们验证了VDACL对应于VDAC的质膜(pl-)亚型的假说。我们检测到pl-VDACL的mRNA表达，与已报道的pl-VDAC序列一致。然而，我们未能通过异源表达重现VDACL通道的表型。接下来，我们测试了线粒体ATP/ADP转运体、苍术糖苷和bongcrekic acid对ATP释放的影响，没有发现一致的效果，因此，该转运体不太可能是VDACL通道的候选分子。较少

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