title: Nitric oxide stimulates NCX1 and NCX2 but inhibits NCX3 isoform by three distinct molecular determinants.
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.
authors.affiliation: Division of Pharmacology, Department of Neuroscience, School of Medicine, University of Naples Federico II, Naples, Italy.
authors.forename: Agnese
authors.initials: A
authors.lastname: Secondo
authors.forename: Pasquale
authors.initials: P
authors.lastname: Molinaro
authors.forename: Anna
authors.initials: A
authors.lastname: Pannaccione
authors.forename: Alba
authors.initials: A
authors.lastname: Esposito
authors.forename: Maria
authors.initials: M
authors.lastname: Cantile
authors.forename: Pellegrino
authors.initials: P
authors.lastname: Lippiello
authors.forename: Rossana
authors.initials: R
authors.lastname: Sirabella
authors.forename: Takahiro
authors.initials: T
authors.lastname: Iwamoto
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Di Renzo
authors.forename: Lucio
authors.initials: L
authors.lastname: Annunziato
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D005434:Flow Cytometry; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms / Q000187:drug effects; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000187:drug effects*
chemical_list: D020033:Protein Isoforms; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D002118:Calcium