Division of Pharmacology, Department of Neuroscience, School of Medicine, University of Naples Federico II, Naples, Italy.
Mol Pharmacol. 2011 Mar;79(3):558-68. doi: 10.1124/mol.110.069658. Epub 2010 Dec 15.
In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in baby hamster kidney cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorometry and patch clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis, and chimera strategies. Our data revealed four main findings. First, the NO-donor S-nitroso-N-acetylpenicillamine (SNAP; 10 nM) and the NO-precursor L-arginine (10 mM) were both able to increase NCX1 activity in a cGMP-independent way. Moreover, within the amino acid sequence 723 to 734 of the f-loop, Cys730 resulted as the target of NO on NCX1. Second, SNAP and L-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In addition, the membrane-permeable 8-bromoguanosine-cGMP alone was able to mimic the stimulatory effect of the gaseous mediator, suggesting the involvement of a cGMP-dependent mechanism. Within the amino acid sequence 699 to 744 of the f-loop, Ser713 was the NO molecular determinant on the NCX2 protein; Third, NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of Cys156 in the α1-region outside the f-loop. Finally, the activity of the two NCX3 chimeras-obtained by the replacement of the NO-insensitive NCX3 region with the homologous NO-sensitive segments of NCX1 or NCX2-was potentiated by SNAP. Together, the present data demonstrate that NO differently regulates the activity of the three gene products NCX1, NCX2, and NCX3 by modulating specific molecular determinants.
在这项研究中，通过单细胞 Fura-2 荧光微测法和膜片钳技术，研究了一氧化氮 (NO) 在单一转染每种亚型的幼仓鼠肾细胞中对 NCX1、NCX2 和 NCX3 交换器活性的调节作用。此外，通过缺失、定点突变和嵌合策略确定了 NO 对每种亚型的分子决定因素。我们的数据揭示了四个主要发现。首先，NO 供体 S-亚硝基-N-乙酰青霉胺 (SNAP; 10 nM) 和 NO 前体 L-精氨酸 (10 mM) 都能够以 cGMP 非依赖性方式增加 NCX1 的活性。此外，在 f 环的 723 到 734 个氨基酸序列中，Cys730 是 NCX1 上的 NO 靶标。其次，SNAP 和 L-精氨酸能够增加 NCX2 的活性，但这种作用被鸟苷酸环化酶抑制剂 1H-[1,2,4]恶二唑[4,3-a]喹喔啉-1-酮 (ODQ) 所阻止。此外，膜通透的 8-溴鸟苷-cGMP 本身能够模拟气态介质的刺激作用，表明涉及 cGMP 依赖性机制。在 f 环的 699 到 744 个氨基酸序列中，Ser713 是 NCX2 蛋白上的 NO 分子决定因素；第三，NO 以 cGMP 非依赖性方式下调 NCX3 的活性。这种 NO 抑制作用发生在 f 环外的 α1 区的 Cys156 水平。最后，通过用 NCX1 或 NCX2 的同源 NO 敏感片段替换 NCX3 的无 NO 敏感区域获得的两种 NCX3 嵌合体的活性被 SNAP 增强。总之，本研究数据表明，NO 通过调节特定的分子决定因素，不同地调节三种基因产物 NCX1、NCX2 和 NCX3 的活性。
