Nitric Oxide Stimulates NCX1 and NCX2 but Inhibits NCX3 Isoform by Three Distinct Molecular Determinants
Nitric Oxide Stimulates NCX1 and NCX2 but Inhibits NCX3 Isoform by Three Distinct Molecular Determinants
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DOI：
10.1124/mol.110.069658
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发表时间：
2011-03
影响因子：
3.6
通讯作者：
A. Secondo;P. Molinaro;A. Pannaccione;A. Esposito;Maria Cantile;P. Lippiello;R. Sirabella;T. Iwamoto;G. di Renzo;L. Annunziato
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文献类型：
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作者：
A. Secondo;P. Molinaro;A. Pannaccione;A. Esposito;Maria Cantile;P. Lippiello;R. Sirabella;T. Iwamoto;G. di Renzo;L. Annunziato
在这项研究中，一氧化氮（NO）的作用，在调制的活性NCX 1，NCX 2，和NCX 3交换机进行了研究，在婴儿仓鼠肾细胞单独转染这些亚型的单细胞Fura-2-显微荧光和膜片钳。此外，每个亚型的NO分子决定簇被确定为缺失，定点突变，嵌合体的战略。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NCX 1活性。此外，在f环的氨基酸序列723至734内，Cys 730作为NCX 1上的NO的靶标。第二，SNAP和l-精氨酸能够增加NCX 2活性，但这种作用被鸟苷酸环化酶抑制剂1H-[1，2，4]恶二唑并[4，3-a]喹喔啉-1-酮（ODQ）阻止。此外，膜可渗透的8-溴鸟苷-cGMP单独能够模拟气体介质的刺激作用，表明cGMP依赖性机制的参与。在f环的氨基酸序列699至744中，Ser 713是NCX 2蛋白上的NO分子决定簇;第三，NCX 3活性以cGMP非依赖性方式被NO下调。这种NO抑制作用在f环外α1区的Cys 156水平发挥。最后，两个NCX 3嵌合体的活性-通过用NCX 1或NCX 2的同源NO敏感片段替换NO不敏感NCX 3区域获得-通过SNAP增强。总之，目前的数据表明，NO通过调节特定的分子决定簇来不同地调节三种基因产物NCX 1、NCX 2和NCX 3的活性。
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.