Sensitivity of the synaptic membrane Na+/Ca2+ exchanger and the expressed NCX1 isoform to reactive oxygen species

Abstract

Two plasma membrane proteins, the Na+/Ca2+ exchanger (NCX) and the Ca2+-ATPase, are major regulators of free intraneuronal Ca2+ levels as they are responsible for extrusion of Ca2+ from the intracellular to the extracellular medium. Because disruption of cellular Ca2+ regulation plays a role in damage occurring under conditions of oxidative stress, studies were conducted to assess the sensitivity of the NCX to reactive oxygen species (ROS). Exchanger activity in brain synaptic plasma membranes and in transfected CHO-K1 cells was inhibited following brief exposure to the peroxyl radical generating azo initiator 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) and to peroxynitrite. Incubation with hydrogen peroxide did not alter NCX activity, even at 800 microM concentration. In CHO-K1 cells transiently transfected with the NCX1 isoform of the exchanger, AAPH treatment decreased the maximal transport capacity (Vmax), whereas the K(act) remained unchanged. Peroxynitrite led to an increase in K(act) with no change in Vmax. Loss of activity following exposure to either AAPH or peroxynitrite was associated with the formation of high molecular weight aggregates of NCX, and AAPH also caused fragmentation of the exchanger protein. These findings suggest that the NCX is sensitive to biologically relevant ROS and could be involved in the loss of Ca2+ homeostasis observed under oxidative stress.

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

“…However, treatment with exogenous H 2 O 2 did not significantly stimulate or inhibit NCX activity in our study (data not shown) and those of others (18,44). The failure of exogenous H 2 O 2 to mimic NCX inhibition by NADH implies that NADH-induced H 2 O 2 generation by the DPI-sensitive enzyme, and the subsequent NCX inhibition, are highly site-specific and finely regulated processes.…”

“…Thus, the modification reaction underlying NCX stimulation remains unresolved. On the other hand, there is evidence showing depressed NCX activity by ROS (17,44). Dixon et al (17) demonstrated that ROS-induced NCX inhibition was prevented by superoxide dismutase and catalase.…”

Oxygen free radicals and redox biology of organelles

“…Many of the proteins affected (mostly inactivated) by oxidation are involved in transmembrane transport of different ions and molecules, for example Na + /Ca 2+ exchanger from synaptic membranes (Huschenbett et al 1998), Ca 2+ -ATPase in alveolar macrophages (Hoyal et al 1996), non-selective cation channels in hepatoma cells (Feranchak et al 2003), and Na + channels in isolated cardiac myocytes (Bhatnagar et al 1990). Lipid peroxidation results in the release of 4-hydroxy-2,3-trans-nonenal (4HN; Benedetti et al 1980), that has numerous damaging effects on membrane proteins, for instance, impairment of glutamate transport in cortical astrocytes (Blanc et al 1998) or inhibition of several neutrophil PM ion transport ATPases (Siems et al 2003).…”

Redox Regulation of Store-Operated Ca2+Entry

“…High cytosolic Ca 2 + promotes Ca 2 + -dependent inactivation of Orai1, dissociation of Orai1 from STIM1 molecules, and STIM1 inactivation (48,105,178). Redox modifications can also regulate Ca 2 + extrusion from cells as reactive thiols have been detected and investigated in both NCX (79,87,161) and PMCA (92,211,212). While more recent studies support the idea of oxidation increasing NCX activity (99) the exact mechanisms remain unclear as cysteine mutations also promote NCX activation (169).…”