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.

Endoplasmic reticulum refilling and mitochondrial calcium extrusion promoted in neurons by NCX1 and NCX3 in ischemic preconditioning are determinant for neuroprotection

“…This effect was exclusively due to NO-dependent NCX1 activation, since it was counteracted by siNCX1 and L-NAME treatments. This finding is in accordance with data recently published by Secondo et al, 18 showing that an increase in NCX1 activity is promoted by NO. Moreover, NCX1-increased activity promotes ER Ca 2 þ refilling, thereby avoiding ER stress in neurons exposed to OGD.…”

Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation

“…[ (19). Briefly, PC12 cells grown on glass coverslips were loaded with 10 M Fura-2/AM for 1 h at room temperature in normal Krebs solution containing 5.5 mM KCl, 160 mM NaCl, 1.2 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM glucose, and 10 mM HEPES-NaOH (pH 7.4).…”

Neuronal NCX1 overexpression induces stroke resistance while knockout induces vulnerability via Akt

“…Furthermore, a lack of highly selective compounds modulating NCX1 activity [14][15][16][17][18] has generated an array of conflicting results on the role of this antiporter in stroke. 6,[19][20][21] We hypothesized that NCX1 might play a relevant role in neuroprotection during brain ischemia since: (1) an increase in NCX1 expression/activity raises Akt phosphorylation, 22 a well-known signaling of neuronal survival [23][24][25] that increases neuronal resistance to brain ischemia; and (2) under anoxic conditions, NCX1 could work in the reverse mode, promoting Ca 2þ -influx and favoring Ca 2þ -refilling into the endoplasmic reticulum (ER), which is depleted under these conditions, thus allowing neurons to delay apoptosis.…”