PT - JOURNAL ARTICLE AU - Agnese Secondo AU - Pasquale Molinaro AU - Anna Pannaccione AU - Alba Esposito AU - Maria Cantile AU - Pellegrino Lippiello AU - Rossana Sirabella AU - Takahiro Iwamoto AU - Gianfranco Di Renzo AU - Lucio Annunziato TI - NITRIC OXIDE STIMULATES NCX1 AND NCX2 BUT INHIBITS NCX3 ISOFORM BY THREE DISTINCT MOLECULAR DETERMINANTS AID - 10.1124/mol.110.069658 DP - 2010 Jan 01 TA - Molecular Pharmacology PG - mol.110.069658 4099 - http://molpharm.aspetjournals.org/content/early/2010/12/15/mol.110.069658.short 4100 - http://molpharm.aspetjournals.org/content/early/2010/12/15/mol.110.069658.full AB - In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in BHK-cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorimetry and patch-clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis and chimera strategies. Our data showed that (1) the NO-donor SNAP (10nM) and the NO-precursor L-arginine (10mM) were both able to increase NCX1 activity in a cGMP-independent way. Moreover, within the amino acid sequence 723-734 of the f-loop, cysteine730 resulted as the target of NO on NCX1; (2) SNAP and L-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor ODQ. In addition, the membrane permeable 8-Br-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 699-744 of the f-loop, serine713 was the NO molecular determinant on the NCX2 protein. (3) NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of cysteine156 in the α1-region outside the f-loop. Finally, (4) 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. Collectively, the present data demonstrated that NO differently regulates the activity of the three gene products NCX1, NCX2 and NCX3 by modulating specific molecular determinants.