Abstract
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.
Footnotes
This work was supported by Cofinanziamento 2008 [Grant COFIN 2008]; and by the Ministero Affari Esteri, Direzione Generale per la Promozione e la Cooperazione Culturale Fondi Italia-Cina, 2008 [Grant Legge 401/1990]; Ministero della Salute, Ricerca Sanitaria Ricerca finalizzata, 2006 [Grant RF-FSL352059]; Ministero della Salute, Ricerca Oncologica 2006; Ministero della Salute, Progetto Strategico, 2007; and Ministero della Salute, Progetto Ordinario, 2007.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.069658.
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ABBREVIATIONS:
- NCX
- Na+-Ca2+ exchanger
- BHK
- baby hamster kidney
- ODQ
- 11H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
- SNAP
- S-nitroso-N-acetylpenicillamine
- AM
- acetoxymethyl ester
- TEA
- tetraethylammonium
- nNOS
- neuronal nitric-oxide synthase
- eNOS
- endothelial nitric-oxide synthase
- DAF-2DA
- 4,5-diaminofluorescein-2-diacetate
- Wt
- wild type
- PKG
- cGMP-dependent kinase
- NMDG+
- N-methyl-d-glucamine+.
- Received October 26, 2010.
- Accepted December 13, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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